Clearance of anthophyllite fibers from the rat lung and the formation of asbestos bodies

ENVIRONMtNTAl

REStARCH

22.

13-21

(1980)

Clearance of Anthophyllite and the Formation A. HOLMES

Received

Fibers from the Rat Lung of Asbestos Bodies AND A. MORGAN

June

I?. 1979

The length distributions of both uncoated and coated fibers in the rat lung were measured at various times up to ? years following exposure to anthophyllite asbestos by inhalation. Fibers were measured by optical microscopy using a membrane filter technique which enables fibers with diameters down to about 0.2 pm to be detected. The frequency of uncoated fibers less than 5 Frn in length decreased from 5 Zc; at 100 days after exposure to 193 after 2 years. These findings confirm and extend earlier investigations which showed that fibers in this length range are cleared more efficiently from the lung than longer fibers. The length distributions of uncoated and coated fibers were dissimilar. No coated fiber
INTRODUCTION

It was reported by Vorwald et al. (1951) that the formation of asbestos bodies occurs much less readily in the rat than in the guinea pig. This finding has been confirmed by Botham and Holt (1972a) who discussed the mechanism of body formation and the reason for the difference between these species. In the present study, length distributions of uncoated and coated fibers in the rat lung were characterized at various times after exposure to anthophyllite asbestos by inhalation. The significance of fiber length in body formation is discussed and also the time scale over which it occurs. Additional information on the effect of fiber length on the clearance of fibers from the lung is included which extends the results obtained in an earlier investigation (Morgan PI ~1.. 1978). MATERIALS

AND METHODS

Animuls. The animals used in this investigation were an inbred strain from the Medical Research Council Radiobiology Unit, Harwell, derived from a nucleus of outbred Alderley Park (Strain 1) SPF rats. At the time of administration they were 5 months old and weighed about 350 g. They were fed on a cubed diet and water nd libitrrm. Administration

of asbestos. The anthophyllite used was the standard reference sample (Timbre11 ef al., 1968) prepared under the auspices of the International Union Against Cancer (UICC). The fibers of this material are relatively thick (Timbre11 et al.. 1970) and virtually all can be resolved with the optical microscope using phase contrast. It was administered to rats by inhalation using “nose only” exposure in the type of chamber described by Evans et ul. (1973). The first three I3 0013-9351/80/030013-09$02.00/0 Copynghf 411 right\

,c 1980 hy Academic Prec,. Inc of reproductmn sn any form reserved

14

HOLMES

AND

MORGAN

rats to be killed were exposed for 30 min only which would have resulted in the deposition of about 20 pg of anthophyllite in the alveolar region. The rat killed after 2 years was exposed for 3 hr on each of 5 consecutive days which would have resulted in the deposition of about 300 Kg of fiber. Mrasurrrnen~ yffibers in lung. When rats were killed the lungs were fixed in buffered formalin. To obtain the fibers for examination the postcaval lobe of the right lung was digested with sodium hypochlorite using the technique described by Morgan and Holmes (1980). In this, fibers are obtained on a membrane filter (Millipore GS, pore size 0.22 pm). Half the filter was placed on a microscope slide and cleared with a dimethylformamideiacetic acid/water mixture (Le Guen, personal communication). The cleared filter was mounted in Neutral Mounting Medium (Gurr). Uncoated fibers (those without any optically visible protein coating) exceeding 2 pm in length were counted and sized under phase contrast with a Zeiss Photomicroscope III using a magnification of about 640 (x 10 eyepiece, x 1.6 Optovar, x40 objective) and Porton-type eyepiece graticule. No doubt fibers <2 pm in length were present but they cannot be detected reliably by optical microscopy. Coated fibers, being much more readily detected, were counted and sized at a lower magnification (about 256). To measure the density (fibers mm-“) of uncoated fibers, continuous scans parallel to the cut edge of the filter were made, until a minimum of 200 fibers had been measured. To measure the density of coated fibers the entire half filter was scanned. loo100

i

730

DAYS

DAYS

F?

so-

/ / i oCL

9

5

-

99

IO-19920-399LO-799 FIBRE

FIG. lfter

I. inhalation

Length

distributions exposure.

of uncoated

and

coated

>a0

LENGTH

,,m

anthophyllite

fibers

in rat lung

at various

times

CLEARANCE

OF

ANTHOPHYLLITE TABLE

CO[INI-M~DIAN RAI LUNG

Rat No. I 2 3 4

LFh(iT-Hs AT V.ZRIOUS

(pm)

01 UNCOATED TIMES FOI LOWING

Uncoated Killed

at

No.

I AND Co-\~tn FIBERS ISOI.,ATED FROM THF EXPOS~RF. IO ANIHOPHYLLITE ASBESTOS

fibers

measured

100 days 247 days 332 days 2 years

15

FIBERS

Coated CML

221 227 227 215

No.

4.7 6.0 6.5 I I.0

fibers

measured

CML

51 58 60 96

52 57 59 59

RESULTS

The uncoated and coated fibers measured in the lungs of each animal were classified into six length categories as follows: ~4.9, 5-9.9, 10-19.9, 20-39.9, 40-79.9, and SO pm. The cumulative distributions were plotted on logarithmic probability paper and the count median lengths (CMLs) obtained. These are given in Table 1 together with the total number of fibers measured in each case. The CMLs of uncoated fibers increased from about 5 pm at 100 days to I1 pm at 2 years. The frequency of fibers <5 pm long in the dust cloud to which the animals were exposed was 79% and in lungs immediately following exposure the value was rather similar (Morgan et ~1.) 1978). In Fig. 1 it can be seen that after 100 days the frequency of fibers <5 pm in length had fallen to 52% and to 19% after 2 years. The frequency of fibers in the 5- to lo-pm range was relatively constant over the same period but all the longer categories increased in frequency. No uncoated fibers exceeding 80 pm in length were detected in any of the animals. 12oor

oL1

I.iLU ‘00

200 TIME

AFTER

ADMINISTRATION.

600

BOO

days

FIG. 3. Ratio of uncoated to coated fibers in the rat lung at various times after thophyllite asbestos. Ratio taking all uncoated fibers (0). Ratio taking uncoated fibers h3).

exposure to an>S pm in length

16

FIG. (X720).

HOLMES

3. Coated

fibers

isolated

from

the

AND

lungs

MORGAN

of rats

exposed

to UICC

anthophyllite

asbestos

CLEARANCE

OF

ANTHOPHYLLITE

FIBERS

17

The CMLs of coated fibers are given in Table 1: all values were between 50 and 60 Frn and, if there is any trend with time, it is not very marked. As can be seen from Fig. 1, very few coated fibers occurred in the lo- to 20-pm range and the greatest frequency was invariably in the 40- to 80-pm category. In general, about 20% of the coated fibers were >80 Frn in length. DISCUSSION

The results of the present experiment, when combined with those of the earlier, confirm that fibers <.5 Frn in length are cleared relatively efficiently from the rat lung. The fact that the frequency of fibers in the 5- to lo-pm range is relatively constant implies that such fibers are also removed, but less efficiently than shorter ones. The present data do not rule out the possibility that fibers > 10 pm in length may be cleared from the lung but, if they are, the process is much less effective than for short fibers. No coated fibers 80 pm in length being coated after 2 years residence in the rat lung is lOO%, the value for fibers in the 40- to 80-pm range is only 7%. For the shorter categories the probability is invariably
18

FIG. 4. Coated fibers isolated amosite. (C) fibers of a synthetic

HOLMES

AND

MORGAN

from the lungs of rats exposed nickel chromium fluoramphibole

to (A) UICC (x720).

crocidolite,

(B) UIC(:

CLEARANCE

OF

ANTHOPHYLLITE

FIBERS

19

after administration of the anthophyllite (upper curve). The value of this ratio declined rapidly during the first year and then much more slowly during the second. This could be taken to imply that most fibers, which ultimately become bodies, are coated during the first year following exposure. However, the fact that short fibers are being cleared from the lung over this period will, of itself, produce a fall in the value of this ratio. To obviate this effect, the ratios were recalculated taking only those uncoated fibers >5 pm in length. The resulting (lower) curve confirms that most bodies are formed during the first year after exposure. Values of this ratio in fibers isolated from human lung are generally much lower, except in cases with asbestosis, where the clearance of short fibers is impaired (Morgan and Holmes, 1980). Some coated anthophyllite fibers isolated from rat lung are shown in Fig. 3. Their morphology and color are quite different to coated fibers isolated by the same technique from guinea pig and human lungs. The coating is invariably discontinuous and frequently occurs in the form of globules, often bizarre in shape; it is very pale yellow (almost white) in color and quite different from the golden yellow-brown characteristic of asbestos bodies encountered in human lung. Possible reasons for the difference in body formation in rats and guinea pigs following inhalation of anthophyllite have been discussed by Botham and Holt (1972a). The same workers (1972b) also studied the formation of bodies in rats and guinea pigs following the inhalation of crocidolite and observed bodies very similar in appearance to those shown in Fig. 3. In Fig. 4 are shown bodies isolated from the lungs of rats exposed by inhalation to fibers of crocidolite, amosite, and synthetic nickel chromium fluoramphibole. These are similar in appearance to those formed on

20

HOLMES

AND

MORGAN

anthophyllite, which suggests that the same mechanism is responsible for coating all types of amphibole fiber. For comparison, bodies formed on anthophyllite in the lungs of guinea pigs are shown in Fig. 5. Here the coating is continuous and the bodies are much closer both in appearance and color to those encountered in human lung. Figure 5 is taken at the same magnification as Figs. 3 and 4 and it is apparent that bodies are formed on much shorter fibers in the guinea pig lung than in that of the rat. This accounts for the much greater frequency of bodies in the former. The difference in the response of these two species to long fibers should be borne in mind when assessing the pathological response to inhaled fibrous materials. Although the appearance of coated fibers in the rat differs from that in the human lung certain features are common to both which suggest that similar factors determine whether or not a fiber becomes coated. These are as follows. (a) No coated fiber < 10 pm in length was found in the rat lung. Pooley (1972) has reported that bodies are rarely found in human lung on fibers < 10 pm long and this has been confirmed in our own work (Morgan and Holmes, 1980). (b) No uncoated fibers >80 pm in length were found in the rat lung and such fibers are rarely encountered in human lung. (c) In the lungs of rats exposed to UICC anthophyllite the maximum frequency of coated fibers occurred in the 40- to 80-pm range. In the human lung, the greatest frequency of coated amphibole fibers generally occurs in the 20- to 40-pm range but cases have been reported in which the greatest frequency occurred in the same range as in the rat.

FIG. (~720).

5. Coated fibers isolated from the lungs of a guinea pig exposed to anthophyllite

asbestos

CLEARANCE

OF

ANTHOPHYLLITE

FIBERS

21

ACKNOWLEDGMENTS The authors gratefully acknowledge the support of the Asbestosis Research Council and of the EEC Environmental Research Programme (Project 277-77-10 ENV-UK). The sample of guinea pig lung from which Fig. 5 was obtained was supplied by Professor P. F. Holt. Department of Chemistry, Reading University.

REFERENCES Botham. S. K., and Holt. P. F. t 1972a). Asbestos body formation in the lungs of rats and guinea-pigs after inhalation of anthophyllite. J. P(rrhr)/. 107, 245-252. Botham. S. K., and Holt. P. F. (1972b). The effects of inhaled crocidolites from Transvaal and North-West Cape mines on the lungs of rats and guinea-pigs. BY;/. J. Et-r,. Pnrllol. 53, 6122620. Evans. J. C.. Evans. R. J.. Holmes. A., Hounam, R. F.. Jones. D. M.. Morgan. A.. and Walsh. M. t 1973). Studies on the deposition of inhaled fibrous material in the respiratory tract of the rat and its subsequent clearance using radioactive tracer techniques. I. UICC crocidolite asbestos. Elli.iYOO. RP.F. 6, 180-201. Morgan, A., and Holmes, A. (1980). Concentrations and dimensions of coated and uncoated asbestos fibres in human lung. Brit. J. Itrci. Med.. in press. Morgan, A.. Talbot, R. J., and Holmes, A. t 1978). Significance of fiber length in the clearance of asbestos fibers from the lung. Brit. J. Id. Med. 35, 146- 153. Pooley. F. D. ( 1972). Asbestos bodies, their formation. composition and character. Enr,iro,r. Rc.\. 5, 3633379. Timbrell, V.. Gilson. J. C.. and Webster. I. (1968). IJICC standard reference samples of asbestos. Ilrr. J. Ctrncrr 3, 406-408. Timbrell, V., Pooley, F., and Wagner, J. C. (1970). Characteristics of respirable asbestos fibers. 1~1 “Proceedings of an International Conference on Pneumoconiosis. Johannesburg 1969” tH. A. Shapiro, Ed.). pp. l20- 125. Oxford Univ. Press. London. Vorwald. A. J., Durkan. T. M.. and Prat., P. C. (1951). Experimental studies of asbestosis. AMA Arch. Ind. Hy,y. Occup. Mrcl. 311. l-43.