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The effect of quartz, administered by intratracheal instillation, on the rat lung. I. The cellular response
INVIRONM~NTAI
RtSARCIi
22,
1~ 12 (1980)
The Effect of Quartz, Administered by lntratracheal Instillation, on the Rat Lung. I. The Cellular Response A. MORGAN,
S. R. MOORES.
A. HOLMES, J. C. EVANS, AND A. BLACK
The r-esponse of the lung to quartz, administered by intratracheat gated using bronchopulmonary lavage. The effect on the recovery and other free cells is described. Other indices investigated included the size distribution of the macrophages, lung. estimated by means of a radioactive the
response
administration. investigated be reported
to 5 mg of quartz
was
N. H. EVANS,
instillation, of alveolar the wet
was investimacrophages weight of lung,
and the total number of free macrophages in the tracer technique. In the first series of experiments measured
at various
times
from
In the second, the effect of amounts of quartz ranging at 24 hr and 7 days. Measurements of lactate dehydrogenase elsewhere.
4 hr to 3 months from
after
0.2 to IO mg was in lavage fluid will
INTRODUCTION
The interaction of toxic dusts with macrophages of both alveolar and peritoneal origin has been studied extensively in \,itro (Beck cf ~1.. 1971; Kaw and Zaidi, 1975; Adamis and Timar, 1976). It is known, however, that mineral dusts adsorb proteins (Morgan, 1974; Desai and Richards, 1978) and pulmonary surfactant (Wallace et NI., 1975) to various extents so that their effect on alveolar macrophages when deposited in the lung is likely to be different to that observed in llitro. both in relative and absolute terms. It was decided therefore, to investigate the possibility of assessing cytotoxicity in ~ti\~j in order to (a) obtain more reliable information than can be obtained from in tvitro test systems and (b) to relate the observations to fibrogenicity so that the results may be used predictively to identify potentially fibrogenic materials. In the present investigation, the effect on the lung of quartz, administered to rats by intratracheal instillation, was studied, Quartz is known to be cytotoxic and is also fibrogenic. The results of two series of experiments are described. In the first, the effect of introducing 5 mg of quartz was investigated at various times from 4 hr to 3 months. In the second, the response to various amounts of quartz was studied after 1 and 7 days. The following parameters were quantified: (a) the wet weight of the lungs; (b) the numbers of alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) recovered by bronchopulmonary lavage: (c) the total number of free AM in the lung: (d) the size distribution of AM. The amount of lactate dehydrogenase recovered in lavage fluid was also measured and the results will be reported elsewhere (Moores rf ctl., 1980). The response to other toxic and nontoxic dusts will be compared with that to quartz at a later date.
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2
MORGAN
MATERIALS
ET
AND
AL.
METHODS
RLAf.S
The animals used were an inbred strain from the Medical Research Council Radiobiology Unit, Harwell, derived from a nucleus of outbred Alderley Park (Strain 1) SPF rats. They were 6 months old and weighed about 400 g. They were fed on a cubed diet and water ad libitum. Adrnit~istratim
of Radioactit-e Particles
All animals used in these experiments were exposed to an aerosol of either ‘“‘Ce-labeled cerium oxide (CeO,) or ‘““Pa-labeled thorium oxide (ThO,) several days before instillation of quartz to enable the total number of free AM in the lung to be estimated. The half lives of ‘-‘Ice and snaPa are 32.5 and 27 days, respectively. Samples of CeO, and ThO, with an MMAD of 2 pm were prepared by water sedimentation and irradiated in the Harwell DID0 reactor (6 x 10” neutrons crn~-” set- ‘) for either 1 day (ThO,) or 1 week (CeO,). The activity of the ‘“‘Ce induced under these conditions is about 20 nCi pg.’ cerium and of the Z33Paabout 70 nCi pg-’ thorium. Groups of eight rats were exposed (nose only) to aerosols of either CeO, or ThO, in the type of chamber described by Evans et al. (1973). The amount of radioactive material deposited in the alveolar region was in the range 1 to 10 pg. After each animal was killed, the amount of radioactive dust in the lungs was measured by y-ray spectroscopy. The amount of radioactive dust recovered from the lungs in each combined wash was determined in the same way. As’ the total number of AM in each combined wash was measured, it was possible to estimate the mean weight of either CeO, or ThO, associated with free macophages recovered from the alveolar spaces. Adnlitlistrcrtion of’ Quurt,:
The material used in these experiments was Dowson and Dobson superfine silica (pure quartz). It was administered by intratracheal instillation. An appropriate amount of quartz was weighed into a vial and sterilized by y-irradiation (Hotspot). Sterile isotonic saline was injected into the vial just before administration, and the suspension was shaken vigorously and sonicated for 30 min. Rats, lightly anesthetized with Halothane (Fluotec MkII Vaporiser, Cyprane Ltd., Keighley, Yorkshire) were placed on a board tilted at an angle of 60” and an Abbocath-T plastic cannula was inserted into the trachea with the aid of a laryngoscope. The tip of the cannula was located midway between the larynx and carina. An aliquot (0.5 ml) of the freshly prepared suspension of quartz was drawn into a syringe and injected into the trachea, followed by 0.1 ml of air, over 1 sec. Rats were revived in an upright position. Saline controls were injected with 0.5 ml of sterile saline only. To study the time course of the reactions produced in the lung two experiments were carried out in which 5 mg of quartz was instilled. In the first, the radioactive dust was ThO, and in the second CeO,. Animals were killed serially between 4 hr and 3 months. The results of these two experiments have been combined and are referred to as the first series. In a second series of experiments to study the doseeresponse relationship, different amounts of quartz were administered
EFFECT
OF
QUARTZ
ON
RA-I-
LUNG:
CELLULAR
RESPONSE
3
ranging from 0.2 to 10 mg. At each dose level three rats were killed after 24 hr and three after 7 days. Similar numbers of saline-instilled rats were killed at the same times. The technique differs only in minor details from that described by Morgan et ul. (1977). Rats were killed by ether anesthesia and exsanguinated by cutting the dorsal aorta. The lungs were dissected out, weighed, and a y-count was made. The trachea was cannulated with the sleeve of an Abbocath-G-14 catheter and the lungs were partially immersed in physiological saline (PS) at 37°C. Eight milliliters of Hank’s balanced salt solution (BSS, Gibco-Biocult) at 37°C was instilled over 30 set and the fluid was withdrawn over 30 set by applying slight suction. This was repeated six times in all and the washes were combined in pairs. The lungs were then washed ten times with 8 ml of PS at 37°C. Before withdrawing the PS from the lungs, they were massaged gently for 30 set to increase the recovery of free cells (Brain, 1971). The PS washes were also combined in pairs. The number of cells present in each combined wash was measured with a Fuchs-Rosenthal hemacytometer. Cells were identified on the basis of size and appearance. Rat alveolar macrophages when rounded up are, with very few exceptions, greater than 10 pm in diameter. A population of smaller cells had a mean diameter of about 9 lrn; unlike AM they varied little in size and lysed more rapidly in the hemacytometer. Preparations of cells were obtained with a Cytospin centrifuge (Shandon Southern Products). Stained preparations of cells obtained in combined washes 1 (BSS) and 5 (PS) confirmed that these were mostly PMN (see Fig. 1). Smaller white cells (mainly lymphocytes) were not included in the hemacytometer counts and generally accounted for less than 1% of the combined numbers of AM and PMN. Finally, the diameters of at least 30 AM were measured using a curtain micrometer eyepiece.
To minimize the effect of variations in animal size, fresh lung weights were expressed as a percentage of the body weight at the time of death. In the first series of experiments the mean weight of the lungs of control animals was 0.45 2 0.04% (SD), a value identical to that reported in earlier experiments (Morgan et crl., 1977). For rats instilled with 0.5 ml of PS, and killed over a similar 3-month period to the controls, the weight was 0.48 t 0.06% which does not differ significantly from the controls. However, there was some evidence of a slight increase in weight at 4 and 24 hr. For rats instilled with 5 mg quartz the mean value was 0.56 i: O.OS%, an increase of 24%’ over the controls. This increase was apparent even in animals killed only 4 hr after administration. The results of the second series were rather different in that there was no significant increase in lung weight after 24 hr at any dose level (see Table 1). After 7 days there did appear to be an increase in lung weight with dose but, relative to the saline-instilled controls, the difference was only significant at the IO-mg level.
4
MORGAN
ET
AL.
a
FIG.
1. Photomicrographs
isolated with physiological instilled with
Effect
of free
the Cytospin centrifuge. saline 29 days previously. 5 mg quartz 4 hr previously.
of Quurtz
cells
recovered
(a) Macrophages (b) Macrophages
on the Recovery
in combined
wash
recovered from and polymorphonuclear
5 (physiological rat
instilled
with leukocytes
saline)
and
0.5 ml of from rat
of Free Cells
In all cases only about 1% of the AM recovered by lavage was accounted for in the BSS washes. Numbers increased in the first PS wash and maximum recoveries were obtained in PS washes 3 and 4 (combined wash 5). The recovery of PMN followed a different pattern with a higher proportion (approximately 20%) being
EFFECT
OF
QUARTZ
ON
RAT
LUNG:
TABLE WET
WEIGHT
OF
LUNGS
OF CON
CELLULAR
I
FROG
AND
RATS”
QUARTZ-INSTILLED
Wet weight Amount of quartz instilled Controls ( 15) Saline-instilled 0.2 mg (3+3) 1 mg (3+3) 5 mg (3+3) IO mg (3+3)
5
RESPONSE
of lungs
(Q body
24 hr
weight) 7 Days
0.45 -c 0.04 0.44 0.44 0.44 0.49 0.47
(3+3)
” No. of animals
i 2 ik t
0.01 0.03 0.02 0.04 0.01
0.41 t 0.01 0.44 k 0.02 0.47 -t 0.03 0.48 +- 0.04 0.54 2 0.02
in parentheses
recovered with BSS. Maximum numbers were obtained in PS washes 1 and 2 (combined wash 4) and subsequently the concentration declined more rapidly than for AM. Typical washout patterns for AM and PMN for a quartz-instilled rat are shown in Fig. 2. Once the peak was reached, numbers recovered in subsequent washes declined exponentially. Differences in the recovery patterns suggest that PMN are less firmly attached than AM to the alveolar wall and the more rapid fall in PMN recovery with PS indicates that a greater proportion of the total may be recovered by lavage.
ps Washes
Combined
Wash
FIG. 2. Recovery of free cells by bronchopulmonary rophages and polymorphonuclear leukocytes recovered after instillation of 5 mg quartz.
Number
’
lavage. Fraction in each combined
of total number wash. Rat killed
of mac14 days
6
MORGAN
ET. AL.
In the first series of experiments the mean recoveries of AM from control and saline-instilled rats were 11.0 and 12.2 x lo”, respectively. The difference is not significant. There was no evidence of even a transient increase in AM recovery following the instillation of 0.5 ml saline. The recovery of AM from quartz-treated rats is shown in Fig. 3. At 4 hr the numbers were less than in the controls but increased thereafter. At 4 days and later times the number of AM recovered was on average about twice that for the controls. In the controls, about 99% of the cells recovered by lavage were AM and the corresponding value for saline instilled rats was 97%. In rats instilled with quartz, however, there was a rapid increase in the number of PMN recovered. As shown in Fig. 3, after 4 hr these outnumbered AM by more than 4: 1 and at 24 hr the ratio was about 7: 1. Subsequently, the numbers declined rapidly to a relatively constant value. Between 2 days and 3 months the numbers of AM and PMN recovered by lavage were rather similar and mostly fell within the range 15 to 25 x 10”. The recoveries of AM and PMN in the second series of experiments following instillation of various amounts of quartz are shown in Fig. 4. After 24 hr there was a decline in the numbers of AM recovered with dose from 13.4 (saline instilled) to q”r I B”
t
Total PMN AM
x 2Lh I
Time
FIG. lary
3. lavage
Numbers at various
of macrophages
and
times
instillation
after
After
x l
0
lnstlllotcon
polymorphonuclear of 5 mg quartz.
of Cluartz.Doys
leukocytes
recovered
by bronchopulmo-
EFFECT
OF
QUARTZ
ON
RAT
LUNG:
CELLULAR
7
RESPONSE
70-
60w p
50-
* = u”
LO-
: I; ;;
30 -
L 2 E 20: lo-
O0
10
5 Amount
FIG,. 4. rats killed total
numher
Numbers of macrophages at 24 hr and 7 days following of free
macrophages
and
in the
of
Quartz
polymorphonuclear the instillation lung
are
lnstllled
,mg
leukocytes of various amounts
included.
Mean
values
recovered of quartz. t
from the Estimates
lungs of of the
1 SD.
8.6 X 10” (10 mg quartz). At the same time, however, there was an increase with dose of PMN reaching a maximum of 61 x 10” with 10 mg quartz. The numbers of PMN obtained with 10 mg did not differ significantly from those with 5 mg. At 7 days, there was an increase in the number of AM recovered relative to the saline-instilled animals. The difference is significant with doses of 5 and 10 mg. On the other hand, the recovery of PMN was invariably less than at 24 hr and the response was more nearly linear with dose. The recoveries of AM and PMN obtained at the 5-mg dose level are in good agreement with values obtained at 24 hr and 7 days in the first series of experiments. lZfft>ct cf Qtturtz
otl tlltj Total Ntrttlhrrs
ot Frrr> AM in the Lung
Measurement of the amount of radioactive dust and of the number of AM in PS washes enabled the mean weight of either CeO, or ThO, per AM to be estimated. This value was relatively constant for different PS washes from the same animal. Because the amount of dust deposited in the alveolar region varied from rat to rat the radioactive dust content of the AM was normalized to a lung burden of 1 pg. In controls, the mean normalized dust content of AM averaged 0.05 x lo-” pg. If the assumption is made that all the radioactive dust was contained within the free AM and that there was little transfer either to the pulmonary lymphatics or to other pulmonary cells, then the total number of free AM would be equal to the reciprocal of this normalized dust content. Ferin and Feldstein (1978) have shown that large doses of dust in the lung can result in a greater fraction being transferred to the hilar lymph nodes. However, measurements of radioactive dust in the hilar lymph nodes in the present experiments indicated that, although there was some
8
MORGAN
ET
AL.
transfer at later times, resulting errors would not be great. Autoradiographs prepared from Cytospin preparations confirmed that radioactive dust particles were virtually all associated with AM. This, together with the fact that no birefringent particles of quartz could be detected in PMN by optical microscopy indicates that they are not actively phagocytic. In the first series the mean total number of free AM in the lungs of controls, estimated in this manner, was 20.1 x 10” and in saline-instilled rats 23.7 x 10”. The difference is significant (P < 0.05). The mean value for rats receiving 5 mg of quartz was 42.5 x IO” or about twice the control value. The total number of free AM did not increase immediately but by 4 days it had reached a value of between two and three times that of the controls and showed little change thereafter. Estimates of total AM in the lungs of rats in the second series of experiments are included in Fig. 4. After 24 hr there was no change in the total numbers of AM, relative to the saline-instilled rats, at any dose level. At 7 days, however, there was a significant increase (P < 0.05) at the I-mg dose level and above. Once again, values obtained at the 5-mg dose level were in good agreement with those obtained at corresponding times in the first series of experiments showing that these effects are quite reproducible. The mean diameter of AM recovered in the PS washes from control rats in the first series of experiments was 12.1 pm. About half the cells fell in the lo- to 11.9~pm range and only about 1% exceeded 16 pm. Similar values were obtained for the saline-instilled animals but the mean diameter of AM from quartz-treated rats was about 13 pm. The mean diameters of AM recovered from each group of rats in the second series of experiments are shown in Fig. 5. For rats instilled with quartz, the mean diameter was generally greater than that found in saline-instilled animals: it can be seen that this is due to a reduction in the frequency of AM in the smaller size range (lo- Il.9 pm) and an increase in the frequency of cells in the 14- to 15.9- and >16-pm categories. The proportion of cells in the 12- to 13.9-pm range stayed relatively constant. The largest cells observed had diameters of 22 to 23 pm. As multinucleate cells were observed in Cytospin preparations these are almost certainly “giant” cells. Although an increase in cell diameter from 12 to 13 pm appears quite small it represents an increase in cell volume of 27%. DISCUSSION
The cellular response of the lung to dust administered by intratracheal instillation was investigated by Brain (1971) who found that the administration of a number of insoluble materials, including coa1 dust, carbon, chrysotile asbestos, iron oxide, and barium sulfate, resulted in an increase in the recovery of free cells. In general, the response was maximal after 1 day but was also elevated, relative to the controls, at 4 hr and 3 days. The present study shows that, in the case of quartz, Brain’s findings were correct with regard to the recovery of free cells but that the response at 4 hr and 1 day is due almost entirely to a large increase in the recovery of PMN. Although the recovery of AM from the alveolar spaces does increase, the response is much slower and does not reach a maximum until 4 days
EFFECT
OF
QUARTZ
ON
RAT
LUNG:
CELLULAR 7 clays
2L Hours
1
5mg Ouartz
8.129?20
/:
i)=133!23
EL
dLb IOmg Quartz
b=13,2f2
'1 1
0
LIIIIL IO- 12- lL16119 139 159 179
b.13
>18
Mocrophage
Frc,. 5. instillation
Size distribution of macrophages of 0.5 ml physiological saline
9
RESPONSE
recovered and various
1221
LIIL lo12- IL- 16119 139 159 179
218
O~ametor.qm
from the lungs of rats amounts of quartz.
at 24 hr and
7 days
after
after administration. In the case of quartz, this increase is maintained for at least 3 months. The radioactive tracer technique employed showed that the greater recovery of AM does reflect a real increase in the numbers present in the alveolar spaces. An increase in the numbers of PMN recovered from the lung by lavage has been reported by Hayes et crl. (1977) who exposed rats to cadmium chloride by inhalation. The PMN response peaked after 3 days while that of the AM, as in the present study, did not reach a maximum until Day 4 or 5. Kavet ef ul. (1978) who exposed hamsters to an iron oxide (Fe,O:,) aerosol, found that the number of pulmonary leukocytes (including PMN) peaked at 24 hr and returned to control levels after 3 days. Numbers of AM recovered also showed a slight increase over the controls at 24 hr. Hayes et (11. (1977) equated the influx of PMN into the alveolar spaces with a “cell injury” phase and the subsequent increase in AM as a “repair phase.” Concomitant EM studies showed that Type I pneumocytes were killed in cadmium-exposed rats and this was followed by a proliferation of Type II cells.
IO
MORGAN
ET
AL.
The results of the present investigation demonstrate that the numbers of PMN recovered at 24 hr provides a very sensitive indicator of the acute effect of quartz on the lung. Administration of only 0.2 mg produced an 1%fold increase in recoverable PMN compared with saline-instilled controls. The fact that the numbers of PMN recovered did not differ significantly after the instillation of 5 and 10 mg of quartz indicates that the smaller dose is sufficient to saturate the response from that volume of the lung into which the quartz stimulus was delivered. After 1 week the numbers of PMN recovered were much reduced, particularly at the lower dose levels, and the response was more nearly linear. Although after 24 hr. there was a fall with increasing dose in the number of AM recovered, estimates of the total numbers of AM in the alveolar spaces indicate that this was due to a reduction in recovery, rather than to a real decline in total numbers. After 1 week there was an increase in the recovery of AM but this was only significant at the 5- and IO-me dose level. However, the total number of AM was significantly increased at the I-mg level. The data are not sufficiently precise to show whether the response is linear with dose at this time. Of the other parameters investigated, changes in lung weight appeared to be relatively insensitive in the range of dose levels studied. Although there was a significant increase in weight after 7 days with IO mg of quartz it was quite small (20%). An almost linear increase in the fresh weight of lung following intratracheal instillation of quartz has been reported by Singh rt ~1. (1977). In that case, however, 50 mg was administered to much smaller animals than were used in the present investigation. An increase in the size of AM following administration of various dusts has been reported by other workers. Kavet ft ~1. (1978) found an increase in the size of AM 24 hr after administration of iron oxide and a similar increase was reported by Camner et (11.(1978) following the administration of nickel dust. The present study showed that the increase in size following the administration of quartz was due to a reduction in the frequency of AM in the lo- to 11.9-pm size range and an increase in the frequency of cells with diameters greater than 14 pm. Examination of Cytospin preparations showed that there was an increase in the number of vacuolated AM in quartz-treated rats. The presence of large vacuolated AM in the alveolar spaces has been reported following the administration of quartz by inhalation (Miller et ~1.. 1978a). These changes in size of the AM are difficult to quantify with precision and appear to vary from animal to animal. As the distribution of dusts administered by intratracheal instillation is relatively inhomogeneous, AM recovered from regions of the lung affected by quartz will be diluted with those from unaffected regions. It is interesting to speculate on the PMN response and, in particular, the reason for the rapid influx of these cells into the alveolar spaces and the almost equally rapid decline to a much lower “equilibrium” level. It has been shown (Morgan et crl., 1977) that the uptake by AM of asbestos fibers deposited in the lung by inhalation is complete within 24 hr of exposure. According to Miller et (11. (1978b) particles of quartz are phagocytosed by AM even more rapidly than asbestos fibers. It seems likely, therefore, that the “injury phase,” with its associated influx of PMN only persists while particles of quartz lie free in the alveolar spaces.
EFFECT
OF
QUARTZ
ON
RAT-
LUNG:
CELLULAR
11
RESPONSE
Once the particles have been taken up by AM the stimulus is considerably reduced. The fact that, after 24 hr, the total number of AM in the alveolar spaces did not differ significantly from that in saline-instilled controls suggests that the AM is remarkably resistant to the cytoxic effect of quartz iw ~*il,o. CONCLUSIONS The present study shows that, of the parameters investigated, the recovery of PMN is much the most sensitive indicator of early damage to the lung. The stimulus which results in abnormal numbers of PMN in the alveolar spaces is maintained for at least 3 months and may be due either to small amounts of free quartz released from dead AM, or to a factor secreted by AM which have been damaged sublethally. It appears, however, that the AM themselves are remarkably resistant to the cytotoxic effects of quartz in \~i\*o and there is no evidence of a dramatic fall in numbers following administration of 10 mg. There is a delayed increase in the total number of free AM in the lungs which can be quantified using the radioactive tracer technique described. More recent work (Moores et ~1.. 1980) on the cellular response to other nonfibrous dusts has indicated that the acute PMN response is nonspecific and may be stimulated by reputedly nontoxic dusts such as titanium dioxide but, in such cases, is not sustained. Thus, the chronic PMN and the (delayed) AM response appear to be better related to cytotoxicity and may provide a better index of fibrogenicity. The involvement of AM in silica fibrogenesis has been discussed by Heppleston and Styles (1967) who claim to have recovered a macrophage fibrogenic factor from AM treated with quartz. More recently, Chvapil (19771 has discussed the role of AM in the stimulation of fibroblasts. ACKNOWLEDGMENTS ‘The authors wish to acknowledge Health. Montreal (Project No. Pneumoconiosis Unit. who provided
281.
the
Adamis.
Effects
support of the Institute They would also like
the
of Occupational to thank Dr.
and Environmental J. C. Wagner. MRC
quartz.
REFERENCES Z. and
Timar.
M. r 19761.
of various
mineral
dusts
on macrophages
O~~W/‘. Etll~ir,,,l. Ilc~~r///r 37, 301 -307. Beck, E. G.. Holt, P. F.. and Masrallah. E. T. t 19711. Effects of chrysotile on macrophage cultures. BKt. ./. /!rt/. .\Ic,tl. 28, l79185. Brain.
.I. D.
(19711.
“Inhaled 200-233.
The
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M. (1977).
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Kr.\.
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6,
12
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ET
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Hayes, J. A., Asvadi, S., Strauss, R. H., and Palmer. K. C. (1977). Alveolar epithelial repair and changes in free airway cell populations following cadmium injury. IN “Proceedings of a Symposium on Pulmonary Macrophage and Epithelial Cells, Richland, Washington. September 1976 (C. L. Sanders, R. P. Schneider, G. E. Dagle, and H. A. Ragan, Eds.). ERDA Symposium Series 43. Heppleston, A. G.. and Styles, J. A. (1967). Activity of a macrophage factor in collagen formation by silica. Nofrrrt, (LrindonJ 214. 521 -522. Kavet, R. I.. Brain, J. D., and Levens, D. J. (1978). Characteristics of pulmonary macrophages lavaged from hamsters exposed to iron oxide aerosols. Lob. Itt~,e.st. 38, 3122319. Kaw. J. L., and Zaidi. S. H. (1975). In i’itro studies on the cytotoxic action of different varieties of asbestos dust on macrophages. Acrtr Pl~urrncrc~d. To.rko/. 36, 225-235. Miller, K., Webster. I.. Handfield, R. I. M.. and Skikne. M. I. (1978a). Ultra structure of the lung of the rat following exposure to crocidolite asbestos and quartz. J. Ptrrltol. 124, 39-44. Miller, K., Handlield, R. I. M., and Kagan. E. (1978b). The effect of different mineral dusts on the mechanism of phagocytosis: A scanning electron microscope study. Ent,irm. Rrs. 15. 1399 154. Moores. S. R.. Black. A., Evans. J. C.. Evans, N. H., Holmes. A., and Morgan. A. (1980). The effect of quartz. administered by intratracheal instillation, on the rat lung. II. The short-term biochemical response. El(i,iror). Rrs.. in press. Moores. S. R.. Sykes, S. E., Morgan, A.. Evans, N., Evans, J. C.. and Holmes, A. The short-term cellular and biochemical response of the lung to toxic dusts: An irl i,ii,o cytotoxicity test. ftr “Proceedings of an International Workshop on the in Vitro Effects of Mineral Dusts, Cardiff. September 1979” (R. C. Brown. M. Chamberlain, R. Davies, and I. P. Gormley. Eds.). Academic Press. New York. In press. Morgan, A. (1974). Adsorption of human serum albumin by asbestiform minerals and its application to the measurement of surface areas of dispersed samples of chrysotile. Eni,iron. RCS. 7. 330-341, Morgan, A., Holmes, A.. and Talbot, R. J. (1977). The fate of inhaled asbestos fibres deposited in the rat lung: A quantitative approach. In “Proceedings of a Symposium on Pulmonary Macrophage and Epithelial Cells, Richland, Washington, September 1976” (C. L. Sanders, R. P. Schneider. G. E. Dagle, and H. A. Ragan, Eds.). ERDA Symposium Series 43. Morgan, A. (1980). Fibre dimensions: Their significance in the deposition and clearance of inhaled fibrous dusts. III “Proceedings of a Conference on Occupational Exposures to Fibrous and Particulate Dust and their Extension into the Environment, Society for Occupational and Environmental Health, Washington, December 1977.” Singh, J., Kaw, J. L., Pandey. S. D.. Viswanathan, P. N.. and Zaidi, S. H. (1977). Amino acid changes and pulmonary response of rats to silica dust. Ent~irorr. Res. 14, 452-462. Wallace, W. E., Headley, L. C.. and Weber, K. C. (1975). Dipalmitoyl lecithin surfactant adsorption by kaolin dust irr r,itro. J. Colloid 1nte~firc.r SC,;. 51, 535-537.
RtSARCIi
22,
1~ 12 (1980)
The Effect of Quartz, Administered by lntratracheal Instillation, on the Rat Lung. I. The Cellular Response A. MORGAN,
S. R. MOORES.
A. HOLMES, J. C. EVANS, AND A. BLACK
The r-esponse of the lung to quartz, administered by intratracheat gated using bronchopulmonary lavage. The effect on the recovery and other free cells is described. Other indices investigated included the size distribution of the macrophages, lung. estimated by means of a radioactive the
response
administration. investigated be reported
to 5 mg of quartz
was
N. H. EVANS,
instillation, of alveolar the wet
was investimacrophages weight of lung,
and the total number of free macrophages in the tracer technique. In the first series of experiments measured
at various
times
from
In the second, the effect of amounts of quartz ranging at 24 hr and 7 days. Measurements of lactate dehydrogenase elsewhere.
4 hr to 3 months from
after
0.2 to IO mg was in lavage fluid will
INTRODUCTION
The interaction of toxic dusts with macrophages of both alveolar and peritoneal origin has been studied extensively in \,itro (Beck cf ~1.. 1971; Kaw and Zaidi, 1975; Adamis and Timar, 1976). It is known, however, that mineral dusts adsorb proteins (Morgan, 1974; Desai and Richards, 1978) and pulmonary surfactant (Wallace et NI., 1975) to various extents so that their effect on alveolar macrophages when deposited in the lung is likely to be different to that observed in llitro. both in relative and absolute terms. It was decided therefore, to investigate the possibility of assessing cytotoxicity in ~ti\~j in order to (a) obtain more reliable information than can be obtained from in tvitro test systems and (b) to relate the observations to fibrogenicity so that the results may be used predictively to identify potentially fibrogenic materials. In the present investigation, the effect on the lung of quartz, administered to rats by intratracheal instillation, was studied, Quartz is known to be cytotoxic and is also fibrogenic. The results of two series of experiments are described. In the first, the effect of introducing 5 mg of quartz was investigated at various times from 4 hr to 3 months. In the second, the response to various amounts of quartz was studied after 1 and 7 days. The following parameters were quantified: (a) the wet weight of the lungs; (b) the numbers of alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) recovered by bronchopulmonary lavage: (c) the total number of free AM in the lung: (d) the size distribution of AM. The amount of lactate dehydrogenase recovered in lavage fluid was also measured and the results will be reported elsewhere (Moores rf ctl., 1980). The response to other toxic and nontoxic dusts will be compared with that to quartz at a later date.
0013-Y351/80/030001-12$02.00/O CopyrIght All right\
C; 1980 hy Academtc Pres. Inc. of reproduct~~ m any form reserved
2
MORGAN
MATERIALS
ET
AND
AL.
METHODS
RLAf.S
The animals used were an inbred strain from the Medical Research Council Radiobiology Unit, Harwell, derived from a nucleus of outbred Alderley Park (Strain 1) SPF rats. They were 6 months old and weighed about 400 g. They were fed on a cubed diet and water ad libitum. Adrnit~istratim
of Radioactit-e Particles
All animals used in these experiments were exposed to an aerosol of either ‘“‘Ce-labeled cerium oxide (CeO,) or ‘““Pa-labeled thorium oxide (ThO,) several days before instillation of quartz to enable the total number of free AM in the lung to be estimated. The half lives of ‘-‘Ice and snaPa are 32.5 and 27 days, respectively. Samples of CeO, and ThO, with an MMAD of 2 pm were prepared by water sedimentation and irradiated in the Harwell DID0 reactor (6 x 10” neutrons crn~-” set- ‘) for either 1 day (ThO,) or 1 week (CeO,). The activity of the ‘“‘Ce induced under these conditions is about 20 nCi pg.’ cerium and of the Z33Paabout 70 nCi pg-’ thorium. Groups of eight rats were exposed (nose only) to aerosols of either CeO, or ThO, in the type of chamber described by Evans et al. (1973). The amount of radioactive material deposited in the alveolar region was in the range 1 to 10 pg. After each animal was killed, the amount of radioactive dust in the lungs was measured by y-ray spectroscopy. The amount of radioactive dust recovered from the lungs in each combined wash was determined in the same way. As’ the total number of AM in each combined wash was measured, it was possible to estimate the mean weight of either CeO, or ThO, associated with free macophages recovered from the alveolar spaces. Adnlitlistrcrtion of’ Quurt,:
The material used in these experiments was Dowson and Dobson superfine silica (pure quartz). It was administered by intratracheal instillation. An appropriate amount of quartz was weighed into a vial and sterilized by y-irradiation (Hotspot). Sterile isotonic saline was injected into the vial just before administration, and the suspension was shaken vigorously and sonicated for 30 min. Rats, lightly anesthetized with Halothane (Fluotec MkII Vaporiser, Cyprane Ltd., Keighley, Yorkshire) were placed on a board tilted at an angle of 60” and an Abbocath-T plastic cannula was inserted into the trachea with the aid of a laryngoscope. The tip of the cannula was located midway between the larynx and carina. An aliquot (0.5 ml) of the freshly prepared suspension of quartz was drawn into a syringe and injected into the trachea, followed by 0.1 ml of air, over 1 sec. Rats were revived in an upright position. Saline controls were injected with 0.5 ml of sterile saline only. To study the time course of the reactions produced in the lung two experiments were carried out in which 5 mg of quartz was instilled. In the first, the radioactive dust was ThO, and in the second CeO,. Animals were killed serially between 4 hr and 3 months. The results of these two experiments have been combined and are referred to as the first series. In a second series of experiments to study the doseeresponse relationship, different amounts of quartz were administered
EFFECT
OF
QUARTZ
ON
RA-I-
LUNG:
CELLULAR
RESPONSE
3
ranging from 0.2 to 10 mg. At each dose level three rats were killed after 24 hr and three after 7 days. Similar numbers of saline-instilled rats were killed at the same times. The technique differs only in minor details from that described by Morgan et ul. (1977). Rats were killed by ether anesthesia and exsanguinated by cutting the dorsal aorta. The lungs were dissected out, weighed, and a y-count was made. The trachea was cannulated with the sleeve of an Abbocath-G-14 catheter and the lungs were partially immersed in physiological saline (PS) at 37°C. Eight milliliters of Hank’s balanced salt solution (BSS, Gibco-Biocult) at 37°C was instilled over 30 set and the fluid was withdrawn over 30 set by applying slight suction. This was repeated six times in all and the washes were combined in pairs. The lungs were then washed ten times with 8 ml of PS at 37°C. Before withdrawing the PS from the lungs, they were massaged gently for 30 set to increase the recovery of free cells (Brain, 1971). The PS washes were also combined in pairs. The number of cells present in each combined wash was measured with a Fuchs-Rosenthal hemacytometer. Cells were identified on the basis of size and appearance. Rat alveolar macrophages when rounded up are, with very few exceptions, greater than 10 pm in diameter. A population of smaller cells had a mean diameter of about 9 lrn; unlike AM they varied little in size and lysed more rapidly in the hemacytometer. Preparations of cells were obtained with a Cytospin centrifuge (Shandon Southern Products). Stained preparations of cells obtained in combined washes 1 (BSS) and 5 (PS) confirmed that these were mostly PMN (see Fig. 1). Smaller white cells (mainly lymphocytes) were not included in the hemacytometer counts and generally accounted for less than 1% of the combined numbers of AM and PMN. Finally, the diameters of at least 30 AM were measured using a curtain micrometer eyepiece.
To minimize the effect of variations in animal size, fresh lung weights were expressed as a percentage of the body weight at the time of death. In the first series of experiments the mean weight of the lungs of control animals was 0.45 2 0.04% (SD), a value identical to that reported in earlier experiments (Morgan et crl., 1977). For rats instilled with 0.5 ml of PS, and killed over a similar 3-month period to the controls, the weight was 0.48 t 0.06% which does not differ significantly from the controls. However, there was some evidence of a slight increase in weight at 4 and 24 hr. For rats instilled with 5 mg quartz the mean value was 0.56 i: O.OS%, an increase of 24%’ over the controls. This increase was apparent even in animals killed only 4 hr after administration. The results of the second series were rather different in that there was no significant increase in lung weight after 24 hr at any dose level (see Table 1). After 7 days there did appear to be an increase in lung weight with dose but, relative to the saline-instilled controls, the difference was only significant at the IO-mg level.
4
MORGAN
ET
AL.
a
FIG.
1. Photomicrographs
isolated with physiological instilled with
Effect
of free
the Cytospin centrifuge. saline 29 days previously. 5 mg quartz 4 hr previously.
of Quurtz
cells
recovered
(a) Macrophages (b) Macrophages
on the Recovery
in combined
wash
recovered from and polymorphonuclear
5 (physiological rat
instilled
with leukocytes
saline)
and
0.5 ml of from rat
of Free Cells
In all cases only about 1% of the AM recovered by lavage was accounted for in the BSS washes. Numbers increased in the first PS wash and maximum recoveries were obtained in PS washes 3 and 4 (combined wash 5). The recovery of PMN followed a different pattern with a higher proportion (approximately 20%) being
EFFECT
OF
QUARTZ
ON
RAT
LUNG:
TABLE WET
WEIGHT
OF
LUNGS
OF CON
CELLULAR
I
FROG
AND
RATS”
QUARTZ-INSTILLED
Wet weight Amount of quartz instilled Controls ( 15) Saline-instilled 0.2 mg (3+3) 1 mg (3+3) 5 mg (3+3) IO mg (3+3)
5
RESPONSE
of lungs
(Q body
24 hr
weight) 7 Days
0.45 -c 0.04 0.44 0.44 0.44 0.49 0.47
(3+3)
” No. of animals
i 2 ik t
0.01 0.03 0.02 0.04 0.01
0.41 t 0.01 0.44 k 0.02 0.47 -t 0.03 0.48 +- 0.04 0.54 2 0.02
in parentheses
recovered with BSS. Maximum numbers were obtained in PS washes 1 and 2 (combined wash 4) and subsequently the concentration declined more rapidly than for AM. Typical washout patterns for AM and PMN for a quartz-instilled rat are shown in Fig. 2. Once the peak was reached, numbers recovered in subsequent washes declined exponentially. Differences in the recovery patterns suggest that PMN are less firmly attached than AM to the alveolar wall and the more rapid fall in PMN recovery with PS indicates that a greater proportion of the total may be recovered by lavage.
ps Washes
Combined
Wash
FIG. 2. Recovery of free cells by bronchopulmonary rophages and polymorphonuclear leukocytes recovered after instillation of 5 mg quartz.
Number
’
lavage. Fraction in each combined
of total number wash. Rat killed
of mac14 days
6
MORGAN
ET. AL.
In the first series of experiments the mean recoveries of AM from control and saline-instilled rats were 11.0 and 12.2 x lo”, respectively. The difference is not significant. There was no evidence of even a transient increase in AM recovery following the instillation of 0.5 ml saline. The recovery of AM from quartz-treated rats is shown in Fig. 3. At 4 hr the numbers were less than in the controls but increased thereafter. At 4 days and later times the number of AM recovered was on average about twice that for the controls. In the controls, about 99% of the cells recovered by lavage were AM and the corresponding value for saline instilled rats was 97%. In rats instilled with quartz, however, there was a rapid increase in the number of PMN recovered. As shown in Fig. 3, after 4 hr these outnumbered AM by more than 4: 1 and at 24 hr the ratio was about 7: 1. Subsequently, the numbers declined rapidly to a relatively constant value. Between 2 days and 3 months the numbers of AM and PMN recovered by lavage were rather similar and mostly fell within the range 15 to 25 x 10”. The recoveries of AM and PMN in the second series of experiments following instillation of various amounts of quartz are shown in Fig. 4. After 24 hr there was a decline in the numbers of AM recovered with dose from 13.4 (saline instilled) to q”r I B”
t
Total PMN AM
x 2Lh I
Time
FIG. lary
3. lavage
Numbers at various
of macrophages
and
times
instillation
after
After
x l
0
lnstlllotcon
polymorphonuclear of 5 mg quartz.
of Cluartz.Doys
leukocytes
recovered
by bronchopulmo-
EFFECT
OF
QUARTZ
ON
RAT
LUNG:
CELLULAR
7
RESPONSE
70-
60w p
50-
* = u”
LO-
: I; ;;
30 -
L 2 E 20: lo-
O0
10
5 Amount
FIG,. 4. rats killed total
numher
Numbers of macrophages at 24 hr and 7 days following of free
macrophages
and
in the
of
Quartz
polymorphonuclear the instillation lung
are
lnstllled
,mg
leukocytes of various amounts
included.
Mean
values
recovered of quartz. t
from the Estimates
lungs of of the
1 SD.
8.6 X 10” (10 mg quartz). At the same time, however, there was an increase with dose of PMN reaching a maximum of 61 x 10” with 10 mg quartz. The numbers of PMN obtained with 10 mg did not differ significantly from those with 5 mg. At 7 days, there was an increase in the number of AM recovered relative to the saline-instilled animals. The difference is significant with doses of 5 and 10 mg. On the other hand, the recovery of PMN was invariably less than at 24 hr and the response was more nearly linear with dose. The recoveries of AM and PMN obtained at the 5-mg dose level are in good agreement with values obtained at 24 hr and 7 days in the first series of experiments. lZfft>ct cf Qtturtz
otl tlltj Total Ntrttlhrrs
ot Frrr> AM in the Lung
Measurement of the amount of radioactive dust and of the number of AM in PS washes enabled the mean weight of either CeO, or ThO, per AM to be estimated. This value was relatively constant for different PS washes from the same animal. Because the amount of dust deposited in the alveolar region varied from rat to rat the radioactive dust content of the AM was normalized to a lung burden of 1 pg. In controls, the mean normalized dust content of AM averaged 0.05 x lo-” pg. If the assumption is made that all the radioactive dust was contained within the free AM and that there was little transfer either to the pulmonary lymphatics or to other pulmonary cells, then the total number of free AM would be equal to the reciprocal of this normalized dust content. Ferin and Feldstein (1978) have shown that large doses of dust in the lung can result in a greater fraction being transferred to the hilar lymph nodes. However, measurements of radioactive dust in the hilar lymph nodes in the present experiments indicated that, although there was some
8
MORGAN
ET
AL.
transfer at later times, resulting errors would not be great. Autoradiographs prepared from Cytospin preparations confirmed that radioactive dust particles were virtually all associated with AM. This, together with the fact that no birefringent particles of quartz could be detected in PMN by optical microscopy indicates that they are not actively phagocytic. In the first series the mean total number of free AM in the lungs of controls, estimated in this manner, was 20.1 x 10” and in saline-instilled rats 23.7 x 10”. The difference is significant (P < 0.05). The mean value for rats receiving 5 mg of quartz was 42.5 x IO” or about twice the control value. The total number of free AM did not increase immediately but by 4 days it had reached a value of between two and three times that of the controls and showed little change thereafter. Estimates of total AM in the lungs of rats in the second series of experiments are included in Fig. 4. After 24 hr there was no change in the total numbers of AM, relative to the saline-instilled rats, at any dose level. At 7 days, however, there was a significant increase (P < 0.05) at the I-mg dose level and above. Once again, values obtained at the 5-mg dose level were in good agreement with those obtained at corresponding times in the first series of experiments showing that these effects are quite reproducible. The mean diameter of AM recovered in the PS washes from control rats in the first series of experiments was 12.1 pm. About half the cells fell in the lo- to 11.9~pm range and only about 1% exceeded 16 pm. Similar values were obtained for the saline-instilled animals but the mean diameter of AM from quartz-treated rats was about 13 pm. The mean diameters of AM recovered from each group of rats in the second series of experiments are shown in Fig. 5. For rats instilled with quartz, the mean diameter was generally greater than that found in saline-instilled animals: it can be seen that this is due to a reduction in the frequency of AM in the smaller size range (lo- Il.9 pm) and an increase in the frequency of cells in the 14- to 15.9- and >16-pm categories. The proportion of cells in the 12- to 13.9-pm range stayed relatively constant. The largest cells observed had diameters of 22 to 23 pm. As multinucleate cells were observed in Cytospin preparations these are almost certainly “giant” cells. Although an increase in cell diameter from 12 to 13 pm appears quite small it represents an increase in cell volume of 27%. DISCUSSION
The cellular response of the lung to dust administered by intratracheal instillation was investigated by Brain (1971) who found that the administration of a number of insoluble materials, including coa1 dust, carbon, chrysotile asbestos, iron oxide, and barium sulfate, resulted in an increase in the recovery of free cells. In general, the response was maximal after 1 day but was also elevated, relative to the controls, at 4 hr and 3 days. The present study shows that, in the case of quartz, Brain’s findings were correct with regard to the recovery of free cells but that the response at 4 hr and 1 day is due almost entirely to a large increase in the recovery of PMN. Although the recovery of AM from the alveolar spaces does increase, the response is much slower and does not reach a maximum until 4 days
EFFECT
OF
QUARTZ
ON
RAT
LUNG:
CELLULAR 7 clays
2L Hours
1
5mg Ouartz
8.129?20
/:
i)=133!23
EL
dLb IOmg Quartz
b=13,2f2
'1 1
0
LIIIIL IO- 12- lL16119 139 159 179
b.13
>18
Mocrophage
Frc,. 5. instillation
Size distribution of macrophages of 0.5 ml physiological saline
9
RESPONSE
recovered and various
1221
LIIL lo12- IL- 16119 139 159 179
218
O~ametor.qm
from the lungs of rats amounts of quartz.
at 24 hr and
7 days
after
after administration. In the case of quartz, this increase is maintained for at least 3 months. The radioactive tracer technique employed showed that the greater recovery of AM does reflect a real increase in the numbers present in the alveolar spaces. An increase in the numbers of PMN recovered from the lung by lavage has been reported by Hayes et crl. (1977) who exposed rats to cadmium chloride by inhalation. The PMN response peaked after 3 days while that of the AM, as in the present study, did not reach a maximum until Day 4 or 5. Kavet ef ul. (1978) who exposed hamsters to an iron oxide (Fe,O:,) aerosol, found that the number of pulmonary leukocytes (including PMN) peaked at 24 hr and returned to control levels after 3 days. Numbers of AM recovered also showed a slight increase over the controls at 24 hr. Hayes et (11. (1977) equated the influx of PMN into the alveolar spaces with a “cell injury” phase and the subsequent increase in AM as a “repair phase.” Concomitant EM studies showed that Type I pneumocytes were killed in cadmium-exposed rats and this was followed by a proliferation of Type II cells.
IO
MORGAN
ET
AL.
The results of the present investigation demonstrate that the numbers of PMN recovered at 24 hr provides a very sensitive indicator of the acute effect of quartz on the lung. Administration of only 0.2 mg produced an 1%fold increase in recoverable PMN compared with saline-instilled controls. The fact that the numbers of PMN recovered did not differ significantly after the instillation of 5 and 10 mg of quartz indicates that the smaller dose is sufficient to saturate the response from that volume of the lung into which the quartz stimulus was delivered. After 1 week the numbers of PMN recovered were much reduced, particularly at the lower dose levels, and the response was more nearly linear. Although after 24 hr. there was a fall with increasing dose in the number of AM recovered, estimates of the total numbers of AM in the alveolar spaces indicate that this was due to a reduction in recovery, rather than to a real decline in total numbers. After 1 week there was an increase in the recovery of AM but this was only significant at the 5- and IO-me dose level. However, the total number of AM was significantly increased at the I-mg level. The data are not sufficiently precise to show whether the response is linear with dose at this time. Of the other parameters investigated, changes in lung weight appeared to be relatively insensitive in the range of dose levels studied. Although there was a significant increase in weight after 7 days with IO mg of quartz it was quite small (20%). An almost linear increase in the fresh weight of lung following intratracheal instillation of quartz has been reported by Singh rt ~1. (1977). In that case, however, 50 mg was administered to much smaller animals than were used in the present investigation. An increase in the size of AM following administration of various dusts has been reported by other workers. Kavet ft ~1. (1978) found an increase in the size of AM 24 hr after administration of iron oxide and a similar increase was reported by Camner et (11.(1978) following the administration of nickel dust. The present study showed that the increase in size following the administration of quartz was due to a reduction in the frequency of AM in the lo- to 11.9-pm size range and an increase in the frequency of cells with diameters greater than 14 pm. Examination of Cytospin preparations showed that there was an increase in the number of vacuolated AM in quartz-treated rats. The presence of large vacuolated AM in the alveolar spaces has been reported following the administration of quartz by inhalation (Miller et ~1.. 1978a). These changes in size of the AM are difficult to quantify with precision and appear to vary from animal to animal. As the distribution of dusts administered by intratracheal instillation is relatively inhomogeneous, AM recovered from regions of the lung affected by quartz will be diluted with those from unaffected regions. It is interesting to speculate on the PMN response and, in particular, the reason for the rapid influx of these cells into the alveolar spaces and the almost equally rapid decline to a much lower “equilibrium” level. It has been shown (Morgan et crl., 1977) that the uptake by AM of asbestos fibers deposited in the lung by inhalation is complete within 24 hr of exposure. According to Miller et (11. (1978b) particles of quartz are phagocytosed by AM even more rapidly than asbestos fibers. It seems likely, therefore, that the “injury phase,” with its associated influx of PMN only persists while particles of quartz lie free in the alveolar spaces.
EFFECT
OF
QUARTZ
ON
RAT-
LUNG:
CELLULAR
11
RESPONSE
Once the particles have been taken up by AM the stimulus is considerably reduced. The fact that, after 24 hr, the total number of AM in the alveolar spaces did not differ significantly from that in saline-instilled controls suggests that the AM is remarkably resistant to the cytoxic effect of quartz iw ~*il,o. CONCLUSIONS The present study shows that, of the parameters investigated, the recovery of PMN is much the most sensitive indicator of early damage to the lung. The stimulus which results in abnormal numbers of PMN in the alveolar spaces is maintained for at least 3 months and may be due either to small amounts of free quartz released from dead AM, or to a factor secreted by AM which have been damaged sublethally. It appears, however, that the AM themselves are remarkably resistant to the cytotoxic effects of quartz in \~i\*o and there is no evidence of a dramatic fall in numbers following administration of 10 mg. There is a delayed increase in the total number of free AM in the lungs which can be quantified using the radioactive tracer technique described. More recent work (Moores et ~1.. 1980) on the cellular response to other nonfibrous dusts has indicated that the acute PMN response is nonspecific and may be stimulated by reputedly nontoxic dusts such as titanium dioxide but, in such cases, is not sustained. Thus, the chronic PMN and the (delayed) AM response appear to be better related to cytotoxicity and may provide a better index of fibrogenicity. The involvement of AM in silica fibrogenesis has been discussed by Heppleston and Styles (1967) who claim to have recovered a macrophage fibrogenic factor from AM treated with quartz. More recently, Chvapil (19771 has discussed the role of AM in the stimulation of fibroblasts. ACKNOWLEDGMENTS ‘The authors wish to acknowledge Health. Montreal (Project No. Pneumoconiosis Unit. who provided
281.
the
Adamis.
Effects
support of the Institute They would also like
the
of Occupational to thank Dr.
and Environmental J. C. Wagner. MRC
quartz.
REFERENCES Z. and
Timar.
M. r 19761.
of various
mineral
dusts
on macrophages
O~~W/‘. Etll~ir,,,l. Ilc~~r///r 37, 301 -307. Beck, E. G.. Holt, P. F.. and Masrallah. E. T. t 19711. Effects of chrysotile on macrophage cultures. BKt. ./. /!rt/. .\Ic,tl. 28, l79185. Brain.
.I. D.
(19711.
“Inhaled 200-233.
The
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Camner, nickel
P.. Johansson. dust. Efir,i,-o,i.
Chvapil.
M. (1977).
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t 1978). fibrosis.
315-325. Ann The adsorption
//I
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I/I
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crocidolite.
A.. and Walsh. M. tract of the rat and Gri,i~o!i.
Kr.\.
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12
MORGAN
ET
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Hayes, J. A., Asvadi, S., Strauss, R. H., and Palmer. K. C. (1977). Alveolar epithelial repair and changes in free airway cell populations following cadmium injury. IN “Proceedings of a Symposium on Pulmonary Macrophage and Epithelial Cells, Richland, Washington. September 1976 (C. L. Sanders, R. P. Schneider, G. E. Dagle, and H. A. Ragan, Eds.). ERDA Symposium Series 43. Heppleston, A. G.. and Styles, J. A. (1967). Activity of a macrophage factor in collagen formation by silica. Nofrrrt, (LrindonJ 214. 521 -522. Kavet, R. I.. Brain, J. D., and Levens, D. J. (1978). Characteristics of pulmonary macrophages lavaged from hamsters exposed to iron oxide aerosols. Lob. Itt~,e.st. 38, 3122319. Kaw. J. L., and Zaidi. S. H. (1975). In i’itro studies on the cytotoxic action of different varieties of asbestos dust on macrophages. Acrtr Pl~urrncrc~d. To.rko/. 36, 225-235. Miller, K., Webster. I.. Handfield, R. I. M.. and Skikne. M. I. (1978a). Ultra structure of the lung of the rat following exposure to crocidolite asbestos and quartz. J. Ptrrltol. 124, 39-44. Miller, K., Handlield, R. I. M., and Kagan. E. (1978b). The effect of different mineral dusts on the mechanism of phagocytosis: A scanning electron microscope study. Ent,irm. Rrs. 15. 1399 154. Moores. S. R.. Black. A., Evans. J. C.. Evans, N. H., Holmes. A., and Morgan. A. (1980). The effect of quartz. administered by intratracheal instillation, on the rat lung. II. The short-term biochemical response. El(i,iror). Rrs.. in press. Moores. S. R.. Sykes, S. E., Morgan, A.. Evans, N., Evans, J. C.. and Holmes, A. The short-term cellular and biochemical response of the lung to toxic dusts: An irl i,ii,o cytotoxicity test. ftr “Proceedings of an International Workshop on the in Vitro Effects of Mineral Dusts, Cardiff. September 1979” (R. C. Brown. M. Chamberlain, R. Davies, and I. P. Gormley. Eds.). Academic Press. New York. In press. Morgan, A. (1974). Adsorption of human serum albumin by asbestiform minerals and its application to the measurement of surface areas of dispersed samples of chrysotile. Eni,iron. RCS. 7. 330-341, Morgan, A., Holmes, A.. and Talbot, R. J. (1977). The fate of inhaled asbestos fibres deposited in the rat lung: A quantitative approach. In “Proceedings of a Symposium on Pulmonary Macrophage and Epithelial Cells, Richland, Washington, September 1976” (C. L. Sanders, R. P. Schneider. G. E. Dagle, and H. A. Ragan, Eds.). ERDA Symposium Series 43. Morgan, A. (1980). Fibre dimensions: Their significance in the deposition and clearance of inhaled fibrous dusts. III “Proceedings of a Conference on Occupational Exposures to Fibrous and Particulate Dust and their Extension into the Environment, Society for Occupational and Environmental Health, Washington, December 1977.” Singh, J., Kaw, J. L., Pandey. S. D.. Viswanathan, P. N.. and Zaidi, S. H. (1977). Amino acid changes and pulmonary response of rats to silica dust. Ent~irorr. Res. 14, 452-462. Wallace, W. E., Headley, L. C.. and Weber, K. C. (1975). Dipalmitoyl lecithin surfactant adsorption by kaolin dust irr r,itro. J. Colloid 1nte~firc.r SC,;. 51, 535-537.