Concentration-dependent respiratory response of guinea pigs to a single exposure of cotton dust

ToxICOLOGYANDAPPLIEDPHARMACOLCCY~&

357-366

(1985)

Concentration-Dependent Respiratory Response of Guinea Pigs to a Single Exposure of Cotton Dust MOHAMMED

A. ELLAKKANI,

YVES C. ALARIE, DIETRICH

A. WEYEL,

AND MERYL H. KAROL’ The To.uicology

Laboratory, Department of Industrial Environmental Public Health, University of Pittsburgh. Pittsburgh,

Received

February

20, 1985: accepted

April

Health Sciences. Graduate Pennsylvania I526 I

School

of

29, I985

Concentration-Dependent Respiratory Response of Guinea Pigs to a Single Exposure of Cotton Dust. (1985). ELLAKKANI, M. A., ALARIE, Y. C., WEYEL, D. A., AND KAROL, M. H. Toxicol. Appl. Pharmacol. 80, 357-366. Eight groups of guinea pigs were exposed to cotton dust at concentrations of 2 to 27 mg/m3. Each exposure was for 6 hr. The pulmonary function of each animal was assessed prior to exposure, following exposure, and 18 hr following exposure. Tidal volume decreased while respiratory frequency increased in a concentrationdependent manner. These changes were amplified when the same measurements were conducted while the animals were breathing an atmosphere containing 10% CO* in 19% 02 and 71% Nz. Greatest response was noted at 18 hr postexposure. The concentration-response effects can be applied to evaluate the respiratory potencies of different cotton dusts grown under a variety of conditions. 0 1985 Academic Press, Inc

We have previously reported the pneumotoxious sources, the acute effect, as measured icity in guinea pigs of respirable particles by a decrease in FEVl , varied by as much as from cotton dust (Ellakkani et al., 1984). a factor of 20. Following a single 6-hr exposure at 20 mg/ The present study was undertaken to dem3, histopathological evaluation of tissues termine if a concentration-response relationtaken 18 hr postexposure revealed an inflamship could be detected in guinea pigs by the matory reaction in the lung. The respiratory pulmonary function tests previously depattern at 18 hr was one of rapid shallow scribed. An animal bioassay, when ultimately breathing. This breathing pattern was further calibrated to the human response, would be exaggerated by challenging the animals with extremely useful in evaluating the potency of 10% CO2 (Wong and Alarie, 1982). The cotton dusts from various sources and in pulmonary reaction has been titled “reflex searching for the causative agent(s) in this restriction” (Haldane ef al., 1918) although complex mixture. some interruption of airflow was also noted. The acute effects of cotton dust on human METHODS volunteers has been described recently by Castellan d al. (1984a,b). The effect was Animals. Male, English smooth haired guinea pigs characterized by a decrease in both forced weighing 380 to 420 g were obtained from Hilltop Lab expiratoxy volume (FEV,) and forced vital Animals, Inc., Scottdale, Pennsylvania. Eight groups of animals (four per group) were exposed to increasing capacity (FVC). With cotton dust from var- concentrations of respirable cotton dust particles. ’ To whom correspondence should be addressed.

Cotton dust exposure. Bulk cotton dust was collected by the condenser waste system from the opening and 357

0041-008X/85

$3.00

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

358

ELLAKKANI

carding areas of a mill as described previously (Ellakkani et al.. 1984). The dust was shipped to the University of Pittsburgh by Cotton Inc., Raleigh, N.C. Once received, it was stored at 10°C until 1 day before use. Respirable particles were released by sonicating the bulk dust with a Pitt No. 3 dust generator as described (Weyel et al., 1984). The sonicator was operated at 60 Hz and 6 V. Air at 5 liter/min entered the bottom of the cylinder and carried small particles and some fibers (rendered airborne by sonication) toward the outlet at the top of the cylinder. The outlet was directed into a settling chamber which consisted of a 40-liter glass aquarium (see Fig. 2, Ellakkani et al., 1984). This design permitted settling of fibers and large particles before the dust entered the primary chamber. When the primary chamber outlet was left open and each individual animal exposure chamber was ventilated at 0.7 liter/min. the airborne dust concentration was around 27 mg/m3. Lower concentrations, down to 2 mg/m3, were achieved by attaching pump number 2 to the outlet of the primary chamber to draw in room air at known rates, and by raising the airflow in each animal exposure chamber to 1.8 liter/min. Exposure concentrations were monitored by placing a polytetrafluoroethylene filter (0.2~pm pore size, Schleicher and Schuell, Inc., Keane N.H.) at the outlet of an animal exposure chamber. Gravimetric measurements were obtained at sampling periods of 40 to 60 min. Four samples were taken during each 6-hr exposure. Particle size distribution was determined with an Andersen miniimpactor attached at port C of the glass exposure chamber. Exposure concentrations and airflows used in this study are given in Table 1.

ET AL. One package containing 20 lb of bulk cotton dust was used throughout this study. For each exposure the dust generator was loaded with 30 g of dust and operated for 1 hr. The dust was then removed by suction and replaced with another 30 g of dust. This procedure assured minimal depletion of small particles from the bulk cotton dust and a stable concentration of respirable particles in the animal exposure chambers. Measurement of tidal volume (Vd, whole body plethysmograph pressure (AP), and respiratory JiiequencJ (f) during air breathing and during 10% CO, challenge. Each of the above measurements was made prior to exposure, immediately following exposure, and at 18 hr postexposure for all animals with the method previously described by Matijak-Schaper et al. (1983). Briefly, each animal was placed in a whole body plethysmograph and fitted with a head chamber. Air or 10% CO2 (in 19% 02. 7 1% N2) flowed continuously through the head chamber at 2 liter/min. 1’r was measured by integrating, with time, the airflow signal measured by a differential pressure transducer (Statham PM-l 5) connected to a Fleisch pneumotachograph attached to the head chamber. The pressure in the whole body plethysmograph, AP, was also measured by a Statham PM- I5 pressure transducer. Calibration was performed as previously described (Matijak-Schaper et al., 1983). Measurements were taken during 10 min when room air was drawn through the head chamber and again 4 min after the 10% CO* mixture was introduced into the head chamber. Values for I;, AP, and f‘were obtained from the average of 10 consecutive breaths while animals were breathing air (VT air), (AP air), and (fair) and while breathing 10% CO2 ( I’r CO& (AP COZ), and (.fCO*).

TABLE 1 AIRFLOW,

DUST CONCENTRATIONS

AND PARTICLE

SIZE FOR COTTON

DUST EXPOSURES

Exposure concentration (m/m’)

Airflow through dust generator (liters/min)

Airflow in primary chamber (liters/min)

Airflow in animal exposure chamber (liters/min)

2.0 t 0.20” 2.9 f 0.13 6.0 f 0.43 8.7 + 0.40 12.0 + 0.40 17.0 f 0.21 20.9 -c 0.42 27.0 + 0.30

5 5 5 5 5 5 5 5

70 48 25 18 12 10 8 5

1.8 1.8 1.8 1.8 1.8 1.8 1.8 0.7

a Average of four samples taken at each exposure concentration, X -C SD. ’ From one determination at each exposure concentration.

Particle size: Aerodynamic mass median diameter (Mm), geometric standard deviation b 3.0, 3.0. 2.9, 2.7, 2.8, 2.9, 3.1. 3.0.

1.4 1.4 1.4 1.4 1.6 1.4 1.5 1.4

COTTON

p-v= loops. Airflow signals (p) as obtained above were digitized at a rate of 256 samples/set. The digitized values were stored on floppy discs of a MINC 11-23 microprocessor. These data were then transmitted via phone lines to the DEC-10 computer of the University of Pittsburgh and displayed on a video terminal with copies made on a Zeta plotter. An example is given in Fig. I. The top signal is li with a horizontal line drawn at zero flow, separating inspiration (upward deflection) from expiration (downward deflection). The middle signal represents tidal volume obtained from digital integration of I? The bottom signal represents AP. The phase relationship between the variables is given by vertical lines drawn at zero flow. From these signals, p--VT. loops were plotted as shown in Fig. I. Statistical analyses. Linear least-squares regression analysis was used to evaluate all concentration-response

4”

t

0

I

I

359

DUST INHALATION

data. Ninety-five percent confidence limits for the true mean value of Y were calculated according to Draper and Smith ( 1966). The relationship between VT and A P was analyzed by linear least-squares regression analysis with 95% confidence limits determined for individual Y values according to Ostle (1963).

RESULTS Eflect of Cotton Dust Inhalation and AP

on fT VT,

Results of cotton dust exposure are presented in Figs. 2 to 4 and Table 2. Respiratory frequency increased in a concentration-de-

”

! i

il

4

4

d

9 t

0t

0

VT

VT

1.00

1.00

i’

i I

0t

A!=

Af’

0

(SEC) I+

-

(SEC)-

FIG. I. Plots of digitized v, Vr, and AP signals recorded prior to exposure. Measurements during air breathing (left side) and during 10% CO, challenge (right side) of airflow (v) (top tracing) with line drawn at zero flow for the first five breaths, separating inspiration, upward deflection, from expiration, downward deflection. Tidal volume (Vr) (middle tracing) was obtained from digital integration of l? Whole-body plethysmographic pressure (AP), bottom tracing. Vertical lines drawn from points of zero flow indicate the phase relationship among the three signals. At the right of the tracings is a V-V, loop for one of the first five breaths. The horizontal line drawn separates inspiration (upward) from expiration (downward). The left hand scales for ri and VT also apply to the v--VT curves.

360

ELLAKKANI

,007

ET AL. JO0-

100z L

so-

POSTEXPOSURE DURING AIR

Y)-

POST EXPOSURE DURING 10%C02

18 HR. POST EXPOSURE DURING 10% CO2

FIG. 2. fduring air breathing and during 10% CO2 challenge measured immediately following a 6-hr cotton dust exposure and 18 hr postexposure. The first data point (open circle) represents the mean (SD) for all 32 animals prior to exposure. The other data points represent the average of 4 animals at each exposure concentration. Lines were fitted by linear regression: 95% confidence limits are shown.

pendent manner when measurements were made while animals were breathing ambient air and while breathing the 10% CO2 mixture (Fig. 2). Responses were maximal at 18 hr postexposure. The response while breathing CO1 was more pronounced than that while breathing ambient air, and there was less variation of responses within each group. During CO2 breathing, the maximum response was close to an increase of 100% over preexposure values and thefC0250 (concentration of dust which resulted in a 50% increase in respiratory rate) was close to the mid range of responses, the area where the 95% confidence limit was narrowest (Fig. 2).

Under these conditions, the potency of dust is best expressed as the fC0250. This value was calculated to be 12.5 mg/m3. VT during air breathing (VT air) and during 10% CO2 challenge ( Vr COz) decreased in a concentration-dependent manner (Fig. 3). Decreases were noted immediately after exposure but the maximum effect was obtained 18 hr postexposure. The effect was more pronounced during 10% CO2 challenge than during air breathing and the variation was least for VT COz at 18 hr postexposure. Accordingly, the potency of the dust can also be expressed as the exposure concentration resulting in a decrease in VT CO2 by 50%

COTTON

361

DUST INHALATION

5;4.

5-

POST EXPOSURE DURING 10%C02

4-

2 25 2

POST EXPOSURE DURING AIR

J-

. 34-

2-

2i I?

.

i \

6

iA

1-

0 6

1

10

5

18 HR. POST FIXPOSURE DURING 10% CO2

4

18HR.

2

POST EXPOSURE DURING AIR

33 s 2

1

0 EXPOSURE

CONdiTRATIOH

(UG,Ms)

1:

EXPOSURE

CONdl!TRATlON

(MG,?“‘)

FIG. 3. Vr during air breathing and during 10% CO2 challenge measured immediately following a 6-hr cotton dust exposure and 18 hr postexposure. The first data point (open circle) represents the X (SD) for all 32 animals prior to exposure. The other data points represent the average of 4 animals at each exposure concentration. Lines were fitted by linear regression; 95% confidence limits are shown.

(Vr CO$O) from preexposure Vr CO*. The Vr CO250 was calculated to be 12.8 mg/m3. Similar to findings with f(C0,) as stated above, Vr CO250 is close to one-half the maximum decrease in Vr COz and located close to the mid range of responses where the 95% confidence intervals are narrowest. This measurement would also be appropriate for expression of the potency of a cotton dust. The results for AP are presented in Fig. 4 and are similar to those obtained for Vr. The exposure concentration which decreased by 50% AP(CO& measured 18 hr postexposure, was calculated to be 15.0 mg/m3.

The similarity of the response for Vr and AP was found over the entire range of exposure concentrations as shown in Fig. 5 and by regression analysis. However, Vr was slightly more sensitive than AP as shown by the calculated 50% response concentrations, i.e., 12.8 for VT vs 15.0 mg/m3 for AP. Since the respiratory response to cotton dust was best evaluated by f COz, VT CO*, and AP CO* at 18 hr postexposure, the results for these parameters are presented in Fig. 6 with percentage change from preexposure values. The concentrations (mg/m’) required to increase or decrease each parameter by 50% were calculated and found to

362

ELLAKKANl

ET AL.

POST EXPOSURE DURING 10% CO2

2-

2 z n Q 2 0

POST EXPOSURE DURING AIR

1.5.

I-

0.5.

s

0.5

O:

10

100

2-

2 (L Q 5 x

1.5 -

T-

o.5;

10

2-

18 HR. POST EXPOSURE DURING AIR

1.5.

O;t

,-

3

\

01

EXPOSURE CONC~RAnON

FIG. 4. AP exposure and animals prior concentration.

(YryuJ)

O.11 EXPOSURE

CONC&RATION(MG/U3)

during air breathing and during 10% CO2 challenge measured immediately after a 6-hr 18 hr postexposure. The first data point (open circle) represents the X (*SD) for all 32 to exposure. The other data points represent the average of 4 animals at each exposure Straight lines were fitted by linear regression and 95% confidence limits.

be: 12.1 for f C02, 12.9 for Vr COz, and 15.9 for AP CO2 . Since at 2 mg/m3 exposure concentration, we observed an increase in AP rather than a decrease (see Fig. 4, lower right quadrant), the value at this concentration was plotted at 0% change. Nonetheless, the calculated 50% response concentration for AP (15.9 mg/m’) is very close to 15.0 mg/m3 found with the non-transformed data (Fig. 4). Evaluation

IO0

of ~-VT Loops

V*(air) decreased and f(air) increased following cotton dust exposure to yield the

typical pattern of “rapid shallow breathing” previously described (Ellakkani et al., 1984). Inspection of V and v-VT loops (Fig. 7) indicated the additional presence of airflow interruption at exposure concentrations of 17 mg/m3 and higher. DISCUSSION The effect of cotton dust exposure on f and VT was found to be dependent on the exposure concentration. Linear relationships were obtained for fCOz and VT CO*. Based on these results, we propose that the potency

Yes Yes Yes

Yes Yes

F(6,24) = 1.07

F(6,24)

= 0.73

Yes

= 2.35 = 1.27 = 2.23

F(6,24) F(6,24) F(6,24)

No Yes Yes Yes Yes No

F(6,24) = 1.88

2.59 0.81 2.39 1.30 2.05 5.42

Result’

ANALYSIS. COEFFICIENTS

= = = = = =

19.6 16.8 17.9 74.0 43.6 132.6

= 164.1 = 39.8

F(1,30) = 227.7 F( 1,30) = 224.9 F(1.30) = 70.3

F(1,30) F(1,30)

F( 1,30) = 24.4

F(1,30) F(1.30) F(1,30) F( I ,30) F(1,30) F(1,30)

Test for slopeb

TESTS FOR LINEARITY AND FOR THE DATA PRESENTED

= = = = = =

0.677 2.022 112.8 0.682 1.933 85.23

+ +

0.141X 0.412X 23.7X 0.23X 0.495x 58.02X

LINES

Y(x) = 1.99 - 0.686X Y(x) = 5.23 ~ 2.04X Y(x) = 140.2 + 51.04X Y(X) = 2.39 - 1.26X Y(X) = 5.92 - 2.99X Y(X) = 112.6 + 99.78X

Y(X) Y(X) Y(X) Y(X) Y(x) Y(X)

Equation

SLOPE OF REGRESSION IN FIGS 2 TO 4

AND

r

-0.64 -0.90 0.75 -0.93 -0.94 0.84

-0.58 -0.6 1 0.57 -0.84 -0.74 0.84

Value using individual responses’

CORRELATION

r

-0.83 -0.96 0.92 -0.97 -0.98 0.97

-0.75 -0.88 0.75 -0.95 -0.88 0.90

response d

group

Value using the mean

n Test for linearity, Ho: The relationship of the individual response vs log concentration is linear (at p < 0.05). yes or no. Table value for j= 2.51. ’ Test for slope, Ho: The slope of this relationship is equal to zero (at p i 0.05). For all cases the slope was found to be significantly different from zero. Table value forJ= 4.17. ‘Correlation coefficients using responses of each animal at eight exposure concentrations, N = 32. d Correlation coefficients using the average response of four animals at eight exposure concentrations, N = 8.

During 10% CO2 challenges AP postexposure Vr postexposure f postexposure AP 18 hr postexposure C; 18 hr postexposure .f 18 hr postexposure

= = = = = =

F(6.24) F(6,24) F(6,24) F(6,24) F(6.24) P(6,24)

During ambient air AP postexposure VT postexposure fpostexposure AP 18 hr postexposure VT 18 hr postexposure / I8 hr postexposure

REGRESSION

Test of linearity. F value found”

LEAST-SQUARES

Measurements made postexposure or 18 hr following exposure for all groups exposed to cotton dust

LINEAR

TABLE 2

2 Y 2 5 F

8 2 9

364

ELLAKKANI

ET AL.

flow can occur due to oscillation of a compliant wall in a straight tube, i.e., spasmodic contraction of bronchial smooth muscle. This

I

OJ 1

2

3

5

5

VT (MO4

FIG. 5. Individual values for VT and AP obtained from 32 animals exposed to cotton dust concentrations of 2 to 27 mg/m3. Values were obtained during 10% CO2 challenge at 18 hr postexposure. The line was fitted by linear least-square regression analysis; 95% confidence intervals are shown.

of cotton dusts can be evaluated in the guinea pig by these parameters. The responses obtained by measurement of Vr directly (by integration of airflow) or indirectly by measurement of AP in the whole body plethysmograph were similar since Vr CO250 and AP CO250 values were comparable. Since AP andfcan be measured rapidly in a whole body plethysmograph without the need for a head chamber (Wong and Alarie, 1982) such measurements during 10% CO2 challenge would be adequate to evaluate the potencies of cotton dust from various sources. Furthermore, since the effect on fwas similar to the effect on V, or AP, the single measurement off during CO* challenge may be adequate to compare the potencies of different cotton dusts. Other measurements can be made at selected high concentrations to further characterize the response but are not necessary for initial evaluation of dust potency. Inspection of I’-Vr loops indicated interruption of airflow during both inspiration and expiration at exposure concentrations above 17 mg/m3. The models of Kim et al. (1983) demonstrate that interruption to air-

VT 18 HR. POST EXPOSURE

FREQUENCY

80

01 1

18 HR. POST EXPOSURE DURING 10%COp

// EXPOSURE

’ /

,, ,,,

CONCE’%4ATION

1

0

(MGM43)

FIG. 6. Percentage change of/“(% increase), F’r (% decrease), and AP (% decrease) from preexposure values. Measurements were made during 10% CO2 challenge at 18 hr postexposure. The straight lines were fitted by linear regression and the 95% confidence limits are given.

COTTON

AIR

AIR

VT

365

DUST INHALATION

VT

VT

1.00 9 0I AP

(SEC)Ly

(SEC1L+

FIG. 7. Plots of digitized I? VT, and AP signals and v-‘-v, curves for the animal depicted in Fig. 1 following cotton dust exposure at 27 mg/m’. Measurements were obtained immediately after exposure (left panels) and at 18 hr postexposure (right panels).

may offer an explanation for the pattern of airflow interruption observed at the higher dust concentrations. In conclusion, the exposure system and respiratory parameters measured permitted evaluation of the pneumotoxicity of cotton dust. This approach can be used to compare various grades of cotton dust and dusts from several sources. The method may also be applied to evaluate other types of dust for their acute or chronic pneumotoxicity since Chvalova et al. (1974) reported that experimental silicosis could be detected in rats by use of the CO2 challenge method. ACKNOWLEDGMENTS The bulk cotton dust was supplied by Dr. P. Sasser, Cotton Incorporated, Raleigh, N.C. This project was

supported under Cooperative Agreement 58-7B30-2-426 between the U.S. Department of Agriculture, Dr. J. Robens, Project Officer, and the University of Pittsburgh. Pulmonary performance evaluation was done under Grant ROl-ES02747 from the National Institute of Environmental Health Sciences.

REFERENCES CASTELLAN, R. M., HANKINSON, J. L., AND OLENCHOCK, S. A. (1984a). Acute human ventilatory response to card-generated dust from cotton representing reference standard cotton. Proceedings of the Eight Cotton Dust Research Conference, Beltwide Cotton Production Research Conference, Atlanta, Georgia, 35-37. CASTELLAN. R. M., OLENCHOCK, S. A., HANKINSON. J. L.. MILLNER, P. D., COCKE, J. B.. BRAGG, K. C.. PERKINS, H. H., AND JACOBS, R. R. (1984b). Acute bronchoconstriction induced by cotton dust: Doserelated responses to endotoxin and other dust factors. Ann. Intern. Med. 101, 157-163. CHVALOVA, M., KUNCOVA. M., HAURANKOVA, J.. AND

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PALECEK, F. (1974). Regulation of respiration in experimental silicosis. Physiol. Bohemoslov. 23, 539547. DRAPER, N. R., AND SMITH, H. (1966). Applied Regression Analysis. Wiley. New York. ELLAKKANI, M., ALARIE, Y., WEYEL, D., MAZUMDAR, S., AND KAROL, M. (1984). Pulmonary reactions to inhaled cotton dust: An animal model for byssinosis. Toxicol. Appl. Pharmacol. 74, 267-284. HALDANE, J. S., MEAKINS, J. C., AND PRIESTLEY, J. G. ( 1918). The Reflex Restriction of Respiration Ajier Mass Poisoning. Report No. 5 of the Chemical Warfare Medical Committee, Chemical Defense Establishment. Porton Down, England. KIM, C. S., BROWN, L. K., LEWARS, G. G., AND SACKNER,M. A. (1983). Deposition of aerosol particles and flow resistance in mathematical and experimental

ET AL. airway models. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 55, 154- 163. MATIJAK-SCHAPER, M., WONG, K. L.. AND ALARIE, Y. (1983). A method to rapidly evaluate the acute pulmonary effects of aerosols in unanesthetized guinea pigs. Toxicol. Appl. Pharmacol. 69, 45 I-460. OSTLE, B. (1963). Statistics in Research. Iowa State Univ. Press, Ames, Iowa. WEYEL, D. A., ELLAKKANI, M., ALARIE, Y.. AND KAROL, M. (1984). An aerosol generator for the resuspension of cotton dust. Toxicol. Appl. Pharmacol. 76, 544547. WONG, K. L., AND ALARIE, Y. (1982). A method for repeated evaluation of pulmonary performance in unanesthetized, unrestrained guinea pigs and its application to detect effects of sulfuric acid mist inhalation. To.uicol. Appl. Pharmacol. 63, 72-90.