Fire and Polymers II - ACS Publications - American Chemical Society

Chapter 22

Environmental Nitrogen Dioxide Exposure Hazards of Concern to the U.S. Army 1

1

Downloaded via TUFTS UNIV on July 11, 2018 at 14:56:30 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

M . A. Mayorga , A. J. Januszkiewicz , and Β. E. Lehnert 1

2

Walter Reed Army Institute of Research, Department of Respiratory Research, Washington, DC 20307-5100 Pulmonary Biology-Toxicology Program, Los Alamos National Laboratory, Los Alamos, NM 87545 2

The U.S. Army is concerned with the health effects of acute, short-duration, high-level nitrogen dioxide (NO ) exposure. A substantial amount of information is known about chronic, low-level NO , however, much is unknown about the effects and the mechanisms of injury induced by acute, high-level NO exposure. The combination of experiments involving rodent and ovine models describe the concentration-dose responses, histological effects, cardiopulmonary changes and cellular effects after NO exposure at rest and with exercise. The effects of high-dose NO exposure and the effects of exercise in a rodent model following NO exposure are described for the first time in these experiments. 2

2

2

2

2

2

High levels of nitrogen dioxide (N0 ) i s of concern to the military in both training and combat scenarios. High levels of N0 may be encountered during the combustion of materials within aircraft, ships, submarines and armored vehicles. Peak levels of N0 above 2000 ppm have been measured in armored vehicles penetrated by high temperature shaped rounds, a type of ammunition. Levels of N0 within enclosures generated by the f i r i n g of one's own weapons may also pose a health r i s k , particularly with today' s r e l a t i v e l y smaller, portable and powerful weapons. Though much i s known about the health effects of low-level N0 exposure, less is known of the effects of high-level N0 exposure. Furthermore, though one could surmise increased N0 toxicity and decreased exercise performance after N0 exposure in an exercising animal or human, the effects of exercise on N0 -induced pulmonary toxicity and the effect of NO exposure on exercise i s also unknown. The United States Army i s one of the world's leaders in the testing of procured 2

2

2

2

2

2

2

2

2

2

This chapter not subject to U.S. copyright Published 1995 American Chemical Society Nelson; Fire and Polymers II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

324

FIRE AND POLYMERS II

and developed weapon systems with regard t o system and human performance, i n c a p a c i t a t i o n , v u l n e r a b i l i t y and survivability. T e s t i n g i s conducted as p a r t of the Army's L i v e F i r e T e s t i n g Program (LFTP). A d d i t i o n a l l y , the U.S. Army Health Hazard Assessment Program (HHAP) i s r e s p o n s i b l e f o r e s t a b l i s h i n g methodology f o r the i d e n t i f i c a t i o n and e l i m i n a t i o n of h e a l t h hazards i n weapon systems and p r o v i d i n g a n a l y s i s of p o t e n t i a l h e a l t h r i s k s a s s o c i a t e d with a given weapon system. The HHAP i s concerned with lower l e v e l - N 0 exposure i n c u r r e d i n t r a i n i n g , whereas, the LFTP i s concerned with h i g h e r - l e v e l N0 exposure encountered i n combat. Many other c o u n t r i e s have adopted the U.S. Army's human i n c a p a c i t a t i o n and the h e a l t h hazard r i s k c r i t e r i a f o r i n j u r y i n c u r r e d through the use of v a r i o u s weapon systems. These c r i t e r i a are based on m o d i f i c a t i o n of p u b l i s h e d c i v i l i a n standards and ongoing t o x i c gas research sponsored by the U. S. Army Medical Research and Development Command. The Los Alamos N a t i o n a l Laboratory and the Department of R e s p i r a t o r y Research, Walter Reed Army I n s t i t u t e of Research conducted a s e r i e s of experiments i n r a t s and sheep t o c h a r a c t e r i z e a dose-response curve, t o i d e n t i f y t h r e s h o l d l e v e l s and determinants of t o x i c i t y , c e l l u l a r responses, and the e f f e c t s of e x e r c i s e a f t e r N0 exposure. T h i s paper p r o v i d e s a summary of these experiments which are d e s c r i b e d i n f u r t h e r d e t a i l elsewhere (1.2). The impact of the r e s u l t s of the Army's b a s i c r e s e a r c h and i t s t e s t i n g of r e a l weapon systems, of b a s i c research, and the establishment of t o x i c i t y c r i t e r i a has l e d t o i n t e r i o r s t r u c t u r a l m o d i f i c a t i o n of v e h i c l e s , the i n s t a l l a t i o n of v e n t i l a t i o n and automatic f i r e e x t i n g u i s h i n g systems, and the establishment of i n d i v i d u a l s o l d i e r face masking g u i d e l i n e s . 2

2

2

EXPERIMENTAL Materials Adult, male, Fischer-344, s p e c i f i c - p a t h o g e n f r e e r a t s were obtained from Sasco, Omaha Nebraska, and Harlan Sprague Dawley, s p e c i f i c - p a t h o g e n - f r e e r a t s were obtained from I n d i a n a p o l i s , Inc. Rats were acclimated, housed two per cage, and provided with standard d i e t and water ad l i b i t u m . Disease-free crossbred ewes (3550 kg) were obtained from Ovine Technologies, Inc. New Hope, PA. Sheep were housed i n indoor cages, two per cage, and provided standard feed and water ad l i b i t u m . The sheep were f a s t e d f o r 24 hr p r i o r t o experiments. N0 Exposure i n Rats. Groups of 8-12 animals weighing an average of 253 g were exposed t o 25, 50, 75, 100, 150, 200, or 250 ppm of N0 f o r 2, 5, 15, or 30 min. In separate experiments, animals were exposed t o 5002000 ppm N0 f o r 1 min. Another group of r a t s , s e r v i n g 2

2

2

Nelson; Fire and Polymers II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

22.

MAYORGA ET A L .

Environmental Nitrogen Dioxide Exposure Hazards

as c o n t r o l s , were exposed t o a i r f o r 30 min. Nitrogen d i o x i d e was generated from d i n i t r o g e n t e t r o x i d e (N 0 ) and mixed with anhydrous a i r i n a mixing chamber. The e x i t i n g N0 was then regulated f o r d e s i r e d c o n c e n t r a t i o n and d i r e c t e d i n t o a 12 p o r t i n h a l a t i o n exposure chamber as shown i n F i g u r e 1. Exposure N0 c o n c e n t r a t i o n s were monitored with a dual channel IR-UV spectrophotometer (Binos I n f i c o n , Leybold-Heraeus, Germany). The c o n c e n t r a t i o n of n i t r i c oxide (NO) i n the exposure mixture was l e s s than 30 ppm. Experimental and c o n t r o l animals were s a c r i f i c e d with sodium p e n t o b a r b i t a l IP a t 4, 8, 24 and 48 h r and the lungs e x c i s e d . The lungs were weighed , i n c l u d i n g an e x c i s e d r i g h t c r a n i a l lobe (RCL). The RCL was s e p a r a t e l y weighed before and a f t e r d r y i n g t o p r o v i d e a wet and dry RCL (RCLWW and RCLDW). The remaining lung was f i x e d with 10% f o r m a l i n i n phosphate b u f f e r e d s a l i n e . The f i x e d lung was sectioned, s t a i n e d w i t h hematoxylin and e o s i n and scored f o r the i n t e n s i t y and d i s t r i b u t i o n of f i b r i n , polymorphonuclear leukocytes (PMN), macrophages and red blood c e l l s i n a l v e o l a r spaces, and type I I pneumocyte h y p e r p l a s i a (Table I ) . Data were s t a t i s t i c a l l y analyzed u s i n g one-way a n a l y s i s of v a r i a n c e and Dunnett's t - t e s t . 2

4

2

2

N 0 Exposure i n E x e r c i s i n g Rats. Male Fischer-344 r a t s , weighing 250-300 g were t r a i n e d f o r 19 days on a t r e a d m i l l a t a 15% grade with d a i l y incremental (5 M/min) i n c r e a s i n g v e l o c i t i e s every 30 sec from 10 M/min t o 60 M/min u n t i l the r a t s were able t o perform a 30 min e x e r c i s e (3.) . The d u r a t i o n of e x e r c i s e used i n a l l e x e r c i s e experiments was 30 min. The r a t s were exposed a t r e s t t o 100 ppm N0 or a i r f o r 15 min. E x e r c i s e was performed a t 1 hr pre-, immediately, 8 o r 24 h r post-exposure. Another c o n t r o l group o f r a t s were r e s t e d p o s t - exposure. Animals were s a c r i f i c e d a t e i t h e r 1, 8, or 24 h r . Groups of animals were e x e r c i s e d twice, immediately and a t 8 hr or a t 24 h r post-exposure followed by necropsy. C o n t r o l animals were exposed t o a i r f o r 15 min and were e i t h e r r e s t e d or e x e r c i s e d and s a c r i f i c e d a t 1, 8, or 24 h r p o s t exposure (Table I I ) . Lung weights were determined and h i s t o l o g i c a l examinations were performed as d e s c r i b e d above. 2

2

Lung Lavage A n a l y s i s of N0 -Exposed Rats. A d u l t male Fischer-344 r a t s (250-300 g) were exposed t o 100 ppm N0 X 15 min as d e s c r i b e d above. Lavage time p o i n t s were 8, 24, 48, 72 and 96 hr post-exposure. Six s e q u e n t i a l lung lavages with 8 ml a l i q u o t s of phosphate b u f f e r e d s a l i n e (PBS) were performed on e x c i s e d lungs. Recovered lavage f l u i d was analyzed f o r t o t a l c e l l count, c e l l d i f f e r e n t i a l , t o t a l p r o t e i n by the Lowry method, and p r o t e i n f r a c t i o n s by i o n exchange h i g h performance l i q u i d chromatography (4). 2

2


325

326


eHUTOFF VALVE METERNQ VALVE TO TEMPERATURE CONTROLLER

I

.

DILUTION

^Sf\~

PERFORATED §AfTL£*

•YPAS3 VALVE8

WATER BATH

TO SCRUBBER8 AND EXHAUST F i g u r e 1· Diagram o f r a t exposure system used t o administer a i r or nitrogen dioxide.



0 = not present 1 = focal (present in an occasional alveolar duct and proximal alveolar structures) 2 = multifocal (present in several alveolar ducts and proximal alveolar structures) 3 = diffuse (present in virtually all alveolar ducts and proximal alveolar structures)

0 = not detected

1 = trace to mild appearance (present in few proximal alveoli)

2 = moderate (present in several proximal alveoli)

3 = high degree (present in many proximal and some more distal alveoli)

4 = high intensity (present in essentially all proximal and more distal alveoli)

Intra-alveolar fibrin

Intra-alveolar PMNs

Intra-alveolar AMs

Intra-alveolar RBCs

Type II pneumocyte hyperplasia

PMNs: Polymorphonuclear Leukocytes AMs: Alveolar Macrophages RBCs: Red Blood Cells

DISTRIBUTION

INTENSITY

LESIONS

2

TABLE I RAT HISTOPATHOLOGICAL SCORING SCHEME FOR N0 INDUCED LUNG INJURY


100 100 100 100 100 100 100 100 100

100 100 100

2a, a a b, b b

3al a2 b

d

Ci

3

2

3

2

0 (control) 0 (control) 0 (control)

la b c

2

N 0 Cone (ppm)

Group

15 15 15

15 15 15 15 15 15 15 15 15

15 15 15

Duration of Exposure (min)

15 15 15

Rest (min)

2

immed & 8 immed & 8 immed & 24

lhr pre lhr pre lhr pre immed immed immed 8 8 24

Exercise in Relation to Exp (hr)

8.5 24 24

8 24 1.5 8 24 8.5 24 24.5

1 8 24

Necropsy Post Exp (hr)

TABLE II RAT EXPERIMENTAL DESIGN FOR COMBINATION N0 EXPOSURE AND EXERCISE

22.

MAYORGA ET AL.


Nose versus Lung N0 Exposure i n Sheep: The sheep were prepared with a c h r o n i c c a r o t i d loop one month p r i o r t o s t u d i e s ( 2 ) . On the day of exposure, the animals were instrumented with venous and a r t e r i a l c a t h e t e r s , a 7French t h e r m o d i l u t i o n c a t h e t e r , a nasotracheal tube (Bivona, Inc., Gary, IN), an airway pressure transducer (4-French, model PR-219, M i l l a r M i c r o - T i p , M i l l a r Instruments, Inc., Houston, TX), and an esophageal transducer (8-French, model PR-346, M i l l a r M i c r o - T i p , M i l l a r Instruments, Inc, Houston, TX). A pneumotachometer ( S e r i e s 3700, Hans-Rudolf Inc., Kansas C i t y , KS) was placed at the proximal end of the nasotracheal tube. Cardiopulmonary measurements i n c l u d e d r e s p i r a t o r y r a t e , t r a c h e a l l a t e r a l pressure, p l e u r a l pressure, t i d a l airway flow, core body temperature, a r t e r i a l blood pressure, r i g h t - s i d e d c a r d i a c chamber pressures, pulmonary a r t e r y wedge pressure and c a r d i a c output. Other measurements obtained were a r t e r i a l and mixed venous blood samples, hemoglobin, methemoglobin and hematocrit. Cardiopulmonary v a r i a b l e s d e r i v e d from the d i r e c t measurements d e s c r i b e d above i n c l u d e d t i d a l volume, transpulmonary pressure, lung r e s i s t a n c e , dynamic lung compliance, systemic and pulmonary v a s c u l a r r e s i s t a n c e , and oxygen consumption (5). Sheep (n=6) were exposed t o e i t h e r 500 ppm N0 or a i r f o r 15 min through a system composed of g l a s s , T e f l o n and T e f l o n - c o a t e d components through a nasotracheal tube or a p l a s t i c modified canine anesthesia mask. Nitrogen d i o x i d e and n i t r o g e n monoxide c o n c e n t r a t i o n s were monitored with a dual beam IR-UV spectrophotometer (Binos I n f i c o n , Leybold-Heraeus, Germany) and a manual d e t e c t o r (model 8014-40OA, Matheson Gas Products). Nitrogen monoxide l e v e l s were l e s s than 1 ppm. Cardiopulmonary measurements were performed at immediately p r e - and post-exposure and at 4 and 24 hr post-exposure. Necropsies were performed at 24 hr and wet t o dry lung weight determined, and gross examination performed. Lungs were f i x e d and prepared f o r h i s t o l o g i c a l examination. S t a t i s t i c a l a n a l y s i s was performed u s i n g one-way and two-way a n a l y s i s of v a r i a n c e f o r e f f e c t s of gas c o n c e n t r a t i o n and e f f e c t s over time, r e s p e c t i v e l y . 2

2

Bronchoalveolar Lavage (BAL) of N0 Exposed Sheep. Experimental and c o n t r o l sheep were prepared and exposed as d e s c r i b e d above except t h a t exposure time was i n c r e a s e d to 20 min and experimental time p o i n t s f o r cardiopulmonary measurements (0, immediately p o s t exposure, 0.5, 6 and 24 hr post-exposure) and BAL (0.5, 6, and 24 hr) were expanded. A d d i t i o n a l l y , a b a s e l i n e BAL was performed 2 wk p r i o r to exposure. Bronchoalveolar lavage was performed i n d i f f e r e n t segments of the r i g h t caudal lobe with 90-cm m o d i f i e d f i b e r - o p t i c bronchoscope (Pentax, model FB-PP10, P r e c i s i o n Instrument Corporation, Orangeburg, N.Y.). 2


329

330


Two-hundred ml, i n a l i q u o t s of, 20-30 ml of s t e r i l e normal s a l i n e were i n s t i l l e d and a s p i r a t e d with a 30ml s y r i n g e . The BAL f l u i d (BALF) volume was recorded, pooled, cooled t o 4 °C, and d i v i d e d i n t o a l i q u o t s f o r f u r t h e r t e s t i n g . Bronchoalveolar lavage f l u i d was analyzed f o r t o t a l c e l l count, c e l l d i f f e r e n t i a l , t o t a l p r o t e i n and albumin (4). Necropsies were performed a t 24 hr, wet/dry lung weight r a t i o s determined, gross p a t h o l o g i c a l examination performed, and t i s s u e prepared f o r h i s t o l o g i c a l examination. RESULTS N 0 Exposure i n Rats. Injury, as assessed by percent change of lung wet weights (LWW), demonstrated no s i g n i f i c a n t i n j u r y a f t e r exposure t o 25, 50, or 75 ppm N0 Χ 2, 5, or 15 min, however, the h i s t o l o g i c a l changes of t r a c e - m i l d , f o c a l a l v e o l a r f i b r i n and moderate Type I I pneumocyte h y p e r p l a s i a were noted a t these c o n c e n t r a t i o n s a f t e r >5 min d u r a t i o n . Figures 2, 3a and 3b demonstrate a general i n c r e a s e i n i n j u r y , as measured by LWW, g e n e r a l i z e d f i b r i n index and g e n e r a l i z e d Type I I pneumocyte h y p e r p l a s i a index at N0 c o n c e n t r a t i o n s above 75 ppm and with i n c r e a s i n g d u r a t i o n of exposure. N0 c o n c e n t r a t i o n appeared t o have a g r e a t e r e f f e c t on t o x i c i t y than d u r a t i o n of exposure. Lung wet weight increased with post-exposure time and plateaued at 24 hr except f o r the 250 ppm N0 exposure group, as shown i n Figure 4. Percent change i n RCLDW c o r r e l a t e d with percent change i n LWW. F i g u r e s 2 and 5 showed t h a t short d u r a t i o n - v e r y high c o n c e n t r a t i o n exposures induced more lung i n j u r y than long duration-low c o n c e n t r a t i o n exposures. 2

2

2

2

2

Lung Lavage F l u i d ( L L F ) A n a l y s i s of N0 -Exposed Rats. Lung lavage f l u i d t o t a l p r o t e i n increased a f t e r exposure t o 15 ppm N0 X 15 min at 8 hr, peaked at 24 hr and returned n e a r l y to b a s e l i n e by 96 hr, as demonstrated i n Figure 6. Lung lavage f l u i d albumin and t r a n s f e r r i n demonstrated a s i m i l a r p a t t e r n of e l e v a t i o n and r e t u r n toward b a s e l i n e as the t o t a l protein. Figure 7 showed t h a t s i g n i f i c a n t PMN a l v e o l a r accumulation d i d not occur u n t i l 24 hr and was maximal at 72 hr, whereas, the maximal i n c r e a s e i n a l v e o l a r macrophages d i d not occur u n t i l 48 hr post-exposure. 2

2

N 0 Exposure i n E x e r c i s i n g Rats. Lung wet weight, extent and s e v e r i t y of a l v e o l a r f i b r i n and RBC e x t r a v a s a t i o n increased a f t e r exposure t o 100 ppm N0 X 15 min followed by e x e r c i s e immediately post-exposure or a t 8 hr. (Figure 8, Table I I I ) . These changes were assessed at a 30 min and p o s t - e x e r c i s e necropsy. However, when r a t s were e x e r c i s e d 8 hr f o l l o w i n g exposure and necropsied 24 hr post-exposure, LWW and h i s t o l o g i c a l changes, with the exception of f i b r i n 2

2


22.

MAYORGA ET AL.

357


750

1500

2250

3000

EXPOSURE EQUIVALENTS : [N0 ppm*min] 2

F i g u r e 2. E f f e c t s of a 5, 15 or 30-minute n i t r o g e n d i o x i d e exposure on r a t lung gravimétries. A s t e r i s k s denote s t a t i s t i c a l s i g n i f i c a n c e (P < 0.05) from c o n t r o l (air-exposed). Redrawn and r e p r i n t e d with permission of E l s e v i e r Science Ireland, Ltd.


331


500

750

1500

2250 3000

1200

500

750

2

1500 2250 3000 EXPSURE EQUIVALENTS : [N0 ppm*min]

357

F i g u r e s 3 a,b. E f f e c t o f n i t r o g e n d i o x i d e exposure on r a t lung a) f i b r i n , 24 hours post exposure, and b) Type I I c e l l h y p e r p l a s i a , 48 hours post exposure. Redrawn and r e p r i n t e d with p e r m i s s i o n o f E l s e v i e r Science I r e l a n d , L t d .

2

EXPOSURE EQUIVALENTS : [N0 ppm*min]

357

S

1300

22.


MAYORGA ET AL.

240 Ν0 (15min)

230 -|

2

220

f2

210

g

200

g Si

190 1 8 0

Ο

25 ppm

·

•

50 ppm

•

100 ppm

Δ

75 ppm

150 ppm

A

200 ppm •

250 ppm

-\

£ Ο

170

Ζ

160

ω

140

150

ξ

130

Χ

120

5?

110 100 90 -

—τ 48

80 24

HOURS POST EXPOSURE

Figure 4. Time course o f e f f e c t o f a 15-minute exposure t o n i t r o g e n d i o x i d e on r a t lung gravimétries. Values represent the means + SE f o r 8-12 animals per group. A s t e r i s k s denote s t a t i s t i c a l s i g n i f i c a n c e (P < 0.05) from c o n t r o l (air-exposed).

.

.

.

.

,

ι

1

I

1

•

2000 χ 1 min"

^

1500x1 miι

;

1000x1 mir ^ ' 5 0 0 χ 1 mit1

• . . . s \ . . . . 0

500

1

1000

1

1

1

ι

1

1500

2000

.

.

.

. "

2500

EXPOSURE EQUIVALENTS (ppm χ time)

Figure 5. E f f e c t o f a 1-minute n i t r o g e n d i o x i d e exposure on r a t lung wet weight (LWW). Each p o i n t represents the mean o f 5-12 animals. Redrawn and r e p r i n t e d with permission o f E l s e v i e r Science Ireland, Ltd.


333

334


0

1

• • • • ι

8

I

I '

24

1

' '

48

1

' ' ' I

72

I I •

• ι • • • • ι 9 6

POST-EXPOSURE TIME (HR)

F i g u r e 6. E f f e c t o f n i t r o g e n d i o x i d e exposure on r a t lung lavage p r o t e i n c o n c e n t r a t i o n over a 4-day post-exposure sampling p e r i o d . Shaded area shows normal range f o r c o n t r o l (air-exposed) r a t s . Each p o i n t r e p r e s e n t s the mean o f 4 animals. Redrawn and r e p r i n t e d with permission of E l s e v i e r Science Ireland, Ltd.

"I

1.e*8

0

8

24

48

72

96

P 0 S T - N 0 EXPOSURE TIMES (HR) 2

F i g u r e 7. E f f e c t o f a 15-minute, 100 ppm n i t r o g e n d i o x i d e exposure on r a t lung lavage a l v e o l a r macrophage (AM) and polymorphonuclear leukocyte (PMN) count over a 4-day sampling p e r i o d . Each p o i n t represents the mean o f 4 animals. Redrawn and r e p r i n t e d with permission o f E l s e v i e r Science Ireland, Ltd.


22.


MAYORGA ET AL.

ο

o o o o o o o o o ο +1 +1 +1 +1 +1 +1 +1 +1 +1

CO Ο

+1

CO

00

co O h M O N O f S O N O O O O O O O O O

ω Ο

S3

Ο

+1

r4

Ο ο

CN ο

+1

+1 +1 +1 +1 +1 +1 +1 +1 +1

+1

p p i n p o q p v o p i o

ρ

oo

r-Ητ-Ηθτ-Ηι—ΐτ-Ηθτ-Ητ-Η

CO

CN

N O V O O N f n v O N N

CO

CO

o o o o o o o o o

ο

r^pppooooTfooco

*o

CNJCO^COCNCN^HCNCO

CO

fi

il c

°

+1 +1 +1 +1 +1 +1 +1 +1 +1

+1

ο

+1 CO

OCO"*COCOCOCO«U-25-30 and >50 ppm f o r small and l a r g e animals, r e s p e c t i v e l y ) N0 have been d e s c r i b e d as a r e s u l t of U.S. Army-sponsored small and l a r g e animal experiments. In the small animal model, a complete concentration-response curve and the e f f e c t of e x e r c i s e on i n j u r y have been d e s c r i b e d . Even at the lower l e v e l s of N0 t e s t e d i n these s t u d i e s (25, 50, and 75 ppm X 5 min), h i s t o l o g i c a l changes of N0 t o x i c i t y were noted. I n j u r y as assessed by LWW and h i s t o l o g i c a l changes i n c r e a s e d with i n c r e a s i n g N0 c o n c e n t r a t i o n and exposure time. Nitrogen d i o x i d e c o n c e n t r a t i o n appeared t o be a more important determinant than d u r a t i o n of exposure. E x e r c i s e p o t e n t i a t e d lung 2

2

2

2


22.

MAYORGA ET A L .


SACRIFICE TIME (HR)

F i g u r e 8. E f f e c t o f post-exposure r e s t o r e x e r c i s e on lung gravimétries from a i r (squares)- o r n i t r o g e n d i o x i d e ( c i r c l e s ) - exposed r a t s . Values represent the means + SE f o r 4-6 animals per group. Note exercise-induced lung damage enhancement i n n i t r o g e n dioxide-exposed r a t s . 120

50 45

J

x

1

1

1

Pre Exposure

Immediate Post Exposure

8 Hours Post Exposure

TIME

F i g u r e 9. E f f e c t o f 15-minute n i t r o g e n d i o x i d e exposure on r a t maximal e x e r c i s e performance, indexed by maximum oxygen consumption. Values represent the means + SE f o r 4-6 animals p e r group. Note c o n c e n t r a t i o n - and time-dependent performance decrement a f t e r exposure.


337


338

26 24 22 20 18 16 14 12 H 10 8 6 4 2 0

INSPIRED MINUTE VENTILATION •

lung only nose only

N0 (500 ppm X 15 min) 2

I

—I

I

Pre Postlhr

4hr

24hr

TIME

RESPIRATORY RATE

•

lung only

•

nose only

N0 (500 ppm X 15 min) 2

I

I

Ί

Pre Post 1 hr

4hr

TIME

24hr

F i g u r e s 10 a,b. E f f e c t o f a 15-minute 500 ppm n i t r o g e n d i o x i d e exposure on a) i n s p i r e d minute v e n t i l a t i o n and b) r e s p i r a t o r y r a t e from lung-only and nose-only exposed sheep over a 24-hour p o s t exposure sampling p e r i o d . Values represent t h e means + SD f o r 4 - 6 animals per group.


22.

MAYORGA ET A L .


0.12

COMPLIANCE

0.10

τ

x

Ε ο

• Ο

0.08 Η

nose-only lung-only

ΝΟ2(500 ppm Χ 15 min)

Φ

ο

0.06

CL

Ε ο Ο

Ο) c

0.04 0.02 0.00

ι Pre

1 Post

1— 1hr

4hr

24hr

Sampling Time F i g u r e s 11 a,b. E f f e c t o f a 15-minute 500 ppm n i t r o g e n d i o x i d e exposure on a) lung r e s i s t a n c e and b) dynamic lung compliance, from lung-only and nose-only exposed sheep over a 24-hour p o s t exposure sampling p e r i o d . Values represent the means ± SD f o r 4-6 animals p e r group. Redrawn and r e p r i n t e d with permission o f E l s e v i e r Science Ireland, Ltd.


339


H

-50

N0

2

• •

Air N0

2

PROTEIN

TIME

1— 6hr 24hr

ω 200

300

Baseline

TIME

F i g u r e s 12 a,b. Bronchoalveolar lavage (BAL) f l u i d a) p r o t e i n and b) albumin, i n sheep exposed t o a i r ( c o n t r o l ) o r 500 ppm n i t r o g e n d i o x i d e f o r 20 minutes. Values represent t h e means + SD f o r 4-6 animals p e r group.

—ι 1/2

(500 ppm X 20 min)

Baseline

H

H

ω 200

250

300

ο

>

3 3

ο


F i g u r e s 13 a,b. B r o n c h o a l v e o l a r lavage f l u i d a) e p i t h e l i a l c e l l and b) macrophage counts, i n sheep exposed t o a i r or 500 ppm n i t r o g e n d i o x i d e f o r 20 minutes. Values r e p r e s e n t the means + SD f o r 4-6 animals per group.

£

w

^

342


i n j u r y . There i s a p e r i o d between the exposure and 24 hr post-exposure during which e x e r c i s e p o t e n t i a t e d i n j u r y , however, t h i s p e r i o d of s u s c e p t i b i l i t y disappeared a f t e r 24 hr. Threshold N0 exposure experiments i n sheep revealed i n c r e a s e s i n airway r e s i s t a n c e and decreases i n lung compliance which was more pronounced i n lung-exposed versus nose-exposed sheep. These changes were maximal at 24 hr. In both animal models, LLF and BALF a n a l y s i s corroborate a pulmonary c a p i l l a r y e n d o t h e l i a l leak mechanism of i n j u r y as manifested by increased t o t a l p r o t e i n and albumin. H i s t o l o g i c a l changes i n both models i n c l u d e an exudative pneumonia with PMN i n f i l t r a t i o n and RBC e x t r a v a s a t i o n and Type II pneumocyte h y p e r p l a s i a . The s i g n i f i c a n c e of t h i s research t o the Army LFTP and HHAP i s evident. I f e x t r a p o l a t i o n s from small and l a r g e animal experiments to humans are made, s e v e r a l c o n c l u s i o n s can be generated. F i r s t l y , m i l i t a r y personnel may experience more N0 t o x i c i t y as the c o n c e n t r a t i o n and d u r a t i o n of exposure i n c r e a s e s . Secondly, m i l i t a r y personnel exposed to h i g h - l e v e l , s h o r t - d u r a t i o n N0 exposure i n combat may be at g r e a t e r r i s k t o N0 lung-induced i n j u r y than personnel exposed t o l o w - l e v e l , longer d u r a t i o n N0 exposure, which c o u l d be encountered i n s e l e c t t r a i n i n g s c e n a r i o s ( f i r i n g from bunkers, enclosures, p a r t i a l l y covered f o x h o l e s ) . T h i r d l y , e x e r c i s e can p o t e n t i a t e N0 toxicity. The l i k e l i h o o d of the need of demanding p h y s i c a l performance i s very high i n both m i l i t a r y t r a i n i n g and combat. Fourthly, N0 lung t o x i c i t y can cause decrements i n e x e r c i s e performance which c o u l d have grave i m p l i c a t i o n s f o r the mission of an i n d i v i d u a l s o l d i e r and t h a t of h i s higher command units. L a s t l y , mouth breathing ( i . e . , d u r i n g e x e r c i s e ) of t o x i n s which bypass the p r o t e c t i v e e f f e c t of the n a s a l passages may be a s s o c i a t e d with increased lung toxicity. I r r e s p e c t i v e of the s c e n a r i o , combat or t r a i n i n g , the t o x i c i t y of N0 must be recognized, weapon systems t e s t e d f o r unacceptable emissions of N0 , armored v e h i c l e s , a i r c r a f t and naval v e s s e l s constructed t o reduce the r i s k of f i r e s , and s a f e t y measures such as v e n t i l a t i o n systems and t r a i n i n g of m i l i t a r y personnel i n the c o r r e c t use of r e s p i r a t o r s must be i n s t i t u t e d . As new research provides i n s i g h t s i n t o the mechanisms of N0 lung i n j u r y , p r o p h y l a c t i c and t h e r a p e u t i c i n t e r v e n t i o n s can be i n i t i a t e d t o f u r t h e r reduce the r i s k of N0 t o x i c i t y . 2

2

?

2

2

?

2

2

2

?

2

ACKNOWLEDGEMENTS: The authors wish t o thank a l l the i n v e s t i g a t o r s and t e c h n i c i a n s from the Department of R e s p i r a t o r y Research, Walter Reed Army I n s t i t u t e of Research and from the Pulmonary Biology-Toxicology Program, Los Alamos N a t i o n a l Laboratory who c o n t r i b u t e d t o t h i s research over the years. Special thanks i s given to Daniel S. Oh f o r g r a p h i c s support.


22.

MAYORGA E T AL.


The views, opinions and/or f i n d i n g s contained i n t h i s r e p o r t are those o f the authors and should not be construed as an o f f i c i a l Department o f the Army p o s i t i o n , p o l i c y o r d e c i s i o n unless so designated by other documentation. In conducting research u s i n g animals, t h e i n v e s t i g a t o r s adhered t o the "Guide f o r the Care and Use o f Laboratory Animals", prepared by the Committee and Use o f Laboratory Animals of the I n s t i t u t e o f Laboratory Animal Resources, N a t i o n a l Research C o u n c i l (NIH) P u b l i c a t i o n NO. 87-23, Revised 1985). LITERATURE CITED 1. S t a v e r t , D.M., and Lehnert, B.E. P o t e n t i a t i o n o f the expression of n i t r o g e n dioxide-induced lung i n j u r y by post exposure e x e r c i s e . Environ Research, 1987; 42,1-13. 2. Januszkiewicz, A.J., Mayorga, M.A. Nitrogen dioxide-induced acute lung i n j u r y i n sheep. T o x i c o l . 1994; 89, 279-300. 3. S t a v e r t , D.M., Lehnert, B.E., Wilson, J.S. E x e r c i s e potentiates nitrogen dioxide toxicity. Toxicoloaist, 1987; 7, A46. 4. Gurley, L.R., Valdez, J.G., London, J.E., D e t h l o f f , L.A., Lehnert, B.E., An HPLC procedure f o r t h e lavage of p r o t e i n s in lung lavage fluid. Anal. Biochem. 1988; 172,465-478. 5. Januszkiewicz, A.J., Snapper, J.R., S t u r g i s , J.W., Rayburn, D.B., Dodd, K.T., Phillips, Y.Y., R i p p l e , G.R., Sharpnack, D.D., Coulson, N.M. and Bley, J.A. P a t h o p h y s i o l o g i c response of sheep t o brief h i g h - l e v e l n i t r o g e n d i o x i d e exposure. I n h a l . T o x i c o l . 1992; 4,359-372. RECEIVED April 20, 1995


343

Fire and Polymers II - ACS Publications - American Chemical Society

Recommend Documents