Toxicology, 5 (1975) 193--199 © Elsevier/North Holland, Amsterdam -- Printed in The Netherlands

EFFECT OF INTRATRACHEAL INJECTION OF MICA DUST ON THE LYMPH NODES OF GUINEA PIGS

RAVI SHANKER, ANAND P. SAHU, RAM K.S. DOGRA and SIBTE H. ZAIDI

Industrial Toxicology Research Centre, Post Box 80, Lucknow (India)

(Received April 24th, 1975) (Accepted July 14th, 1975)

SUMMARY

Histopathological changes in the tracheo-bronchial lymph nodes were studied up to 365 days in guinea pigs following intratracheal injection of a suspension of mica dust. In general, the cytotoxic effect provoked by dust was not pronounced as the majority of the swollen dust-laden macrophages retained their normal structure at the termination of the experiment and fibrotic lesions were limited to the formation of thick reticulin fibers. The poor fibrogenic response of mica dust has been at tr ibuted to its low cyto- toxicity.

INTRODUCTION

Earlier survey of mica mines of Bihar province in India revealed a high incidence of pneumoconiosis in workers handling mica [ 1,2]. Studies dealing with the biological effects of a muscovite variety of Indian mica on ex- perimental animals were recently initiated at this Centre [3]. The present communicat ion describes preliminary histological changes encountered in the tracheo-bronchial lymph nodes of guinea pigs as a result of intratracheal inoculation of muscovite mica dust.

METHODS

The source and particle size of mica dust used in the present experiment were the same as those presented in an earlier paper [3]. 40 female guinea pigs of the I.T~R.C. animal house with an average body weight of 300 g were used. 36 animals were inoculated intratracheally with a sterilized dust suspension of 75 mg mica dust in 1.5 ml normal saline, and 4 animals served

193

as saline controls. All the animals were maintained on stock laboratory diet {Hindustan Lever Pellet), leafy vegetables and water ad lib.

The animals were sacrificed at intervals between 24 h and 365 days post- inoculation and autopsied. The tracheo-bronchial lymph nodes were carefully dissected out and fixed in formol saline. After routine fixation the blocks were embedded in paraffin, sections cut at 5 # and stained with hematoxylin and eosin and silver-impregnated for reticulin [4] . Stained sections were examined under ordinary as well as polarized light for assessing cellular response, stromal proliferation and dust concentrat ion in the field.

R E S U L T S

Macroscopically the tracheo-bronchial lymph nodes of guinea pigs, which received an intratracheal injection of mica dust did not reveal any abnormality till 30 days. The nodes at 60 days, however, appeared slightly enlarged and at following periods up to 365 days became prominent and hard to the touch.

Microscopically no significant reaction was seen in the lymph nodes at 24 h, though at 7 days there was scattering of birefringent particles of mica in the subcapsular sinus and cortical zone. At places the particles were corn-

Fig. 1. Lymph node at 30 days. Showing birefringent dust particles under polarised light. × 200 .

194

Fig. 2. Lymph node at 60 days. Medullary sinus contained swollen dust-laden maerophages. × 640.

pletely phagocytosed by macrophages. In addition, there was an increase in the number of germinal centres which contained degenerated mononuclear cells together with dust particles. The changes observed at 15 days did not differ more than at the previous stage except that there was proliferation of mononuclear cells in the cortical region. At the end of 30 days the cortex revealed small foci of mica and pigment-laden macrophages along with some extracellular deposits of birefringent particles (Fig. 1). The medullary sinuses also contained at places swollen dust-laden macrophages which showed a moderate to marked degree of degenerative changes.

There was no further progress in the tissue reaction at 60 days, the majority of swollen dust-laden macrophages present in the cortical zone and medullary sinuses retained their normal structure and were packed with birefringent particles (Fig. 2}. Such areas exhibited infiltration of eosinophils. In general, the lymphocyt ic populat ion had a greater density and there was a slight plasma cell reaction. At 90 days, the periphery of dust areas showed mitotic activity, fibroblastic proliferation and infiltration of macrophages and lymphocytes (Fig. 3). There was, however, very little evidence of cytotoxici ty . The density of birefringent particles varied from place to place when exam-

195

Fig. 3. Lymph node at 90 days. Showing mitotic stage and fibroblastic reaction. × 1600.

ined under polarised light. On silver impregnation the dust foci revealed a loose network of reticulin fibers.

At 120 days the dust-filled macrophages were packed densely in the cortico-meduUary zone and there was a marked proliferation of macrophages. At places the dust foci exhibited marked degeneration of macrophages. Later, some of the dust-laden macrophages showed a foamy appearance at 150 days though their nuclei stained normal ly . On silver impregnation the cortico-meduUary zone revealed moderately thickened reticulin fibers. The reaction at 180 days closely resembled the previous stage.

There was an overall increase in the density of birefringent particles in the nodes at 300 days in the cortico-medullary zone leaving behind thin strips of lymphocytes while at other places there appeared marked proliferation of lymphocytes and fibroblasts. The dust areas showed infiltration of eosino- phils which were degenerated. A significant finding at 330 days was that the swollen dust-laden macrophages retained their normal structure and were associated with the formation of thick reticulin fibers though the density of birefringent particles remained high (Figs. 4--6). At termination of the experiment (365 days) the fibrogenic response in the lymph nodes did not show any further enhancement. Under polarised light masses of birefringent particles representing mica accumulated in the cortico-medullary zone as

196

Fig. 4. Lymph node at 330 days. Dust-laden macrophages retained normal structure. × 640.

Fig. 5. Lymph node at 330 days. Thick reticulin fibrosis. X 160. 197

Fig. 6. Lymph node at 330 days, Showing increased density of birefringent dust particles under polarised light, x 200.

well as in the medulla. The cyto toxic effect produced by mica remained restricted a t this period.

No changes of pathological significance were seen in the lymph nodes of control animals.

DISCUSSION

From the present studies it is evident that there was slow transport of mica dust to the tracheo-bronchial lymph nodes and the majority of swollen dust-laden macrophages retained their normal histological structures even at the end of the experiment. The cyto toxic effect was, however, not pro- nounced and fibrotic lesions consisted o f thick reticulin fibers only. Generally, the more cy to toxic the dust the greater is the likelihood of it becoming extraceUular and the greater is the probabil i ty of it being transpqrted from the dust depo t to be deposited in the lymph nodes [5] . Quartz du~t which is cy to toxic has been found to be deposited in the lymph nodes at a greater rate than an inert dust like ti tanium dioxide [6--8] . When inoculated in animals quay. z produces at first a typical reaction in the lymph nodes followed by collagenous fibrosis [9- -12] . The effect of mica dust on the

198

l y m p h nodes is a l t oge the r d i f f e r en t f r o m t h a t o f quar tz , which could be exp la ined b y its low solubi l i ty values o b t a i n e d in in v i t ro s tudies [ 1 3 ] . Since the so lubi l i ty o f dus t is c losely re la ted to pa thogenes i s o f silicosis or si l icatosis [ 1 4 ] , i t seems obv ious t h a t the p o o r f ibrogenic r esponse of mica dus t obse rved in the p re sen t s t udy cou ld poss ib ly be re la ted to its l ow c y t o t o x i c i t y .

ACKNOWLEDGEMENTS

T h e au tho r s are gra tefu l t o Mr. M.M. Lal fo r the p r e p a r a t i o n o f dus t , to Mrs. S. Khanna , Mr. Lalji Shukla , Mr. S.N. Sr ivastava for technica l he lp and Mr. Musleh A h m a d fo r p h o t o m i c r o g r a p h y .

REFERENCES

1 H. Heimann, S. Moskowitz, C.R.H. Iyer, M.N. Gupta and N.S. Mankiker, Arch. Industr. Hyg. Occup. Med., 8 (1953) 420.

2 M.N. Gupta, Report No. 12, Government of India, Ministry of Labour, New Delhi, 1956.

3 J.L. Kaw and S.H. Zaidi, Exp. Pathol., 8 (1973) 224. 4 H. Gordon and H.H. Sweets Jr., Amer. J. Pathol., 12 (1936) 545. 5 P. Gross and T. Hatch, Int. Arch. Gewerbepathol., 19 (1962) 660. 6 W. Klosterkotter and G. Bunemann, Arch. f. Hyg. u. Bakteriol., 143 (1959) 112. 7 L. le Bouffant, Inhaled Particles and Vapours, Pergamon, Oxford, 1962. 8 G. Nagelschmidt, E.S. Nelson, E.J. King, D. Attygalle and M. Yoganathan, Amer.

Med. Ass. Arch. Industr. Health, 16 (1957) 188. 9 H.W. Schlipkoter, Arch. Environ. Health, 21 (1970) 181.

10 H.W. Schlipkoter, W. Hilscher, F. Pott and E.J. Beck, Inhaled Particles, III, Vol. 1, Unwin, Old Woking, Surrey, 1971.

11 W. Hilscher and H.W. Schlipkoter, Ann. N.Y. Acad. Sci., 200 (1972) 166. 12 R. Shanker, W. Hilscher and G. Gauss, Ind. J. Industr. Med., 20 (1974) 118. 13 Q. Rahman, M.U. Beg, P.N. Viswanathan and S.H. Zaidi, Environ. Physiol. Biochem.,

3 (1973) 281. 14 S.H. Zaidi, Experimental Pneumoconiosis, Johns Hopkins Press, Baltimore, 1969.

199