Aim. occvp. Hyt- Vol. 18, pp. 173-177. Pergunon Preti 1975. Printed in Great Britain
THE INDUCTION OF POLYKARYOCYTES BY VARIOUS FIBROUS DUSTS AND THEIR INHIBITION BY DRUGS IN RATS N. MANOJLOVIC*
Abstract—U.I.C.C. chrysotile, glass fibres, and fibrous quartz were intraperitoneally administered to rats using the cover slip method. The animals were sacrificed at 24-hr intervals and the cover slips prepared for phase contrast and interference microscopy. On the first days mononuclear dendritic and epitheloid macrophages were observed, which had phagocytosed fibrous material and deposited it perinuclearly in lysosomes. Between the fourth and fifth day polykaryocytes appeared, which showed variable sizes and secondary integration of mononuclear cells. Giant cell morphology and mode of development were independent of the type of fibre administered. Control animals which had received Docrentrupper quartz, powdered glass or physiological saline, showed only mononuclear cellular reaction during eight days of the experiment. Animals which had received U.I.C.C. crocidolite were treated with meprobamate and procaine hydrochloride on the third to the fifth day. In this case no polykaryocytes were found. The mechanism of polykaryocyte induction and the influence of the drugs as regards inhibition of polykaryocyte development are discussed.
INTRODUCTION
IN AN earlier report we have described the development and morphology of polykaryocytes following intraperitoneal administration of U.I.C.C. crocidolite to rats (SETHI et al., 1974). In the present investigation, the morphology of polykaryocytes induced by U.I.C.C. chrysotile, glass fibres, and fibrous quartz have been compared under similar experimental conditions. In addition, the influence of intraperitoneallyadministered procaine and meprobamate on the development of polykaryocytes induced by crocidolite has been studied. MATERIALS As experimental dusts U.I.C.C. chrysotile, glass fibre, fibrous quartz and, for the drug effect, U.I.C.C. crocidolite were employed. The glass fibre and fibrous quartz samples ranged in length from 1 to 20 /im and in diameter from 0-25 to 1 fim. Substances used for control groups were Doerentruper quartz (DQ 12, ROBOCK, 1973) powdered glass, and pyrogen-free physiological saline. Both DQ 12 and the powdered glass had particle sizes < 3 /im. Procaine hydrochloride and meprobamate (2-methyl-2-n-propyl-l,3-propanedioldicarbamate) were prepared as TyTode's solutions in a concentration of 10 mg/ml. • Med. Institut fur Lufthygjene und Silikoseforschung an der Universitat Dusseldorf. t Institut fur Hygiene im Zentrum fttr Okologie der Justus-Liebig-Universitat Giessen (BRD). Please address requests for reprints to Prof. Beck, Hygiene-Institut der Universitat, 6300 Giessen, Friedrichstr. 16, BRD. 173
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S. SETHI,* E. G. BECK! and
174
S. SETHI, E. G. BECK and N. MANOJLOVIC
METHODS
Procaine (LD 30 = 25 mg/100 g body wt. rat) 6 rats: five 5 mg doses per day, on days 3, 4 and 5 4 rats: five 5 mg doses per day, on days 4 and 5 6 rats: five 7 -5 mg doses per day on days 3, 4 and 5 4 rats: five 7-5 mg doses per day, on days 4 and 5. Meprobamate (LD J 0 = 41 mg/100 g body wt. rat) 6 rats: five 6 mg doses per day, on days 3, 4 and 5 4 rats: five 6 mg doses per day, on days 4 and 5 6 rats: five 12 mg doses per day, on days 3, 4 and 5 4 rats: five 12 mg doses per day, on days 4 and 5. All animals were killed on the sixth day and cover slips were taken out and processed for phase and interference contrast microscopy. RESULTS Groups treated with fibres only The cellular reaction observed during the first three days following administration of three different fibre types was identical. One side of the cover slips was covered with omental tissue and large deposits of the substance administered, and the other side was covered with a monolayer of mononucleated cells mainly consisting of epitheloid and dendritic macrophages. Mostly the dust was lying within lysosomes which were perinuclearly arranged and only a few fibres were seen lying free in the cytoplasm (Fig. 1). After administration of fibrous quartz, the cells often showed necrobiotic alterations of variable degree, particularly those which were heavily packed with fibres. The cellular reaction after the third day following the administration of dusts differed in the various animal groups: In the animals which had received glass fibres or chrysotile, polykaryocytes became visible on the fourth day. Many round and oval nuclei with prominent nucleoli were lying in groups within the cell. The cytoplasm was mostly homogeneous and lacked structures except for the occasional presence of glass fibres and the vacuolation in the neighbourhood of the nuclei. The cellular membrane appeared thickened due to its infolding and high membranous activity.
Downloaded from http://annhyg.oxfordjournals.org/ at The University of British Colombia Library on June 21, 2015
A total of 136 female Wistar rats was employed. Three groups of 24 animals each were given U.I.C.C. chrysotile A, glass fibres, and fibrous quartz respectively. Three rats of each group were sacrificed at intervals of 24 hr from the first to the 8th day. Three control groups of 8 rats each received glass powder, DQ 12 quartz and physiological saline (2 ml/rat) respectively. An animal of each group was sacrificed at intervals of 24 hr from the first to the 8th day. All dusts were administered peritoneally in doses of 5mg/rat, using the cover slip method. After sacrificing the animals, implanted cover slips were taken out and prepared for microscopical examination as described by SETHI et al (1974). Forty rats received crocidolite on day 1 and were then divided into two groups and treated with procaine or meprobamate respectively by peritoneal injection as follows:
(facing page 174)
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FIG. 1. 48 hr. Dendritic and epitheloid macrophages. Arrows indicate glass fibres lying in lysosomes arranged perinuclearly, with a few fibres lying free in the cytoplasm. (a) Phase contrast 1 240 x . (b) Interference contrast 1 240 x . FIG. 2. 5 days. Polykaryocytes induced by glass fibres with variable cytoplasmic and nuclear morphology, (a) Groups of nuclei (thick arrow) are surrounded by numerous vacuoles (thin arrow) Homogeneous, structureless cytoplasm contains engulfed fibrous material (short arrow), (b) Groups of darker and lighter nuclei are randomly distributed in the cell (arrows). Phase contrast 1 240 x .
Downloaded from http://annhyg.oxfordjournals.org/ at The University of British Colombia Library on June 21, 2015
FIG. 3. 5 days. Secondary incorporation of mononuclear cells by polykaryocytes induced by glass fibres, (a) Several incorporated cells lie in vacuoles within a polykaryocyte (arrows). These cells surround another incorporated cell which is merging with the cytoplasm of the giant cell. Nearby a nucleus is free in the cytoplasm with vacuoles. (b) Incorporated cells in a vacuole within a polykaryocyte and already integrated nuclei (arrows). Phase contrast 1 240 x . FIG. 4. 5 days. Strands of nuclei (arrows) stretching from the periphery to the deeper regions of a polykaryocyte induced by glass fibres. These nuclei are probably derived from secondarily integrated cells, (a) Interference contrast 530 x . (b) Phase contrast 1 240 x. FIG. 5. 6 days. No development of polykaryocytes after simultaneous treatment with crocidolite and meprobamate. Only dendritic and epitheloid macrophages are seen. Phase contrast 1 240 x .
Induction of polykaryocytes
175
Control groups In the groups treated with DQ 12 or powdered glass the early cellular reaction from the first to the third day was similar to that seen in the experimental groups. The mononuclear dendritic and epitheloid macrophages were heavily packed with administered dust. After saline administration the cells were highly vacuolated. Epitheloid cells were rare compared with the experimental groups. Mononuclear cells persisted until the end of the experiment. The only alteration observed was the enlargement of the lysosomes—the typical cytotoxic effect of DQ 12. Meprobamate and procaine groups On the sixth day, all the animals regardless of dose type and duration of drug administration showed a similar cellular reaction.
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On the fifth day, the polykaryocytes formed large syncytia, showing variable cytoplasmic and nuclear morphology at different regions. Groups of darker nuclei lying in the vicinity of darker areas of plasma and groups of lighter nuclei were randomly distributed in the cell. The cytoplasm became vacuolated, homogeneous without showing structures, or contained fibrous material (Fig. 2). The presence of single undamaged cells within the polykaryocytes was striking. These cells seemed to be engulfed secondarily. A mononuclear cell was first seen lying within a concavity formed by the cytoplasmic pseudopodia, being gradually taken up by the polykaryocytes and then lying inside a vacuole within the polykaryocyte (Fig. 3). Starting from one end, the cytoplasm of the incorporated cell finally merged with the main plasm. This process continued until the integrated cell lost its identity and only the nucelus could be made out. Sometimes strands of nuclei stretching from the periphery to the deeper regions were observed. These nuclei probably belonged to the secondarily incorporated cells being pushed deeper into the polykaryocyte as numerous cells were incorporated from the periphery (Fig. 4). All these characteristic symptoms developed until the end of the 8th day of the experiment. Except for the necrobiotic alteration in the cells, development and appearance of polykaryocytes were essentially the same in the animals receiving fibrous quartz as in those receiving glass fibre. Polykaryocytes containing a few nuclei at first appeared on the fourth day and showed a continuous increase in size and number of nuclei until the 8th day. Cellular morphology was also the same as that seen in animals after glass fibre application. The proximity of mononuclear and multinuclear cells and their cytoplasmic extensions was very striking, as well as the demonstration of morphologically-different areas within a single polykaryocyte. This process represents polykaryocyte formation being mediated by the process of fusion. Polykaryocytes became visible on the sixth day following administration of chrysotile fibres. Polykaryocytes at this stage contained only a few nuclei arranged in rosette form within a highly-vacuolated cytoplasm. On the 7th and 8th day, large polykaryocytes containing large numbers of nuclei (often more than 50) were observed. Other cytoplasmic and nuclear characteristics were similar to those in polykaryocytes induced by glass fibres. An important feature of these polykaryocytes was the fact that while most cells joining the main syncytial mass were mononuclear, some already contained two or three nuclei.
176
S. S m n ,
E. G. BECK and N. MANOJLOVIC
The cell population consisted of mononuclear dendritic and epitheloid macrophages which contained phagocytosed crocidolite asbestos fibres lying within lysosomes. Most of the cells were highly vacuolated. Occasionally some cells were rounded up and these showed no vacuolation. No polykaryocytes were observed in any group, regardless of dose and duration of administration of meprobamate and procaine hydrochloride (Fig. 5).
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DISCUSSION This method, which involves implantation of glass cover slips into the rat peritoneal cavity along with experimental dusts, followed by their removal at indicated intervals, allows constant in vitro observations of events induced in vivo (SETHI et al., 1974). Except for the different time intervals at which polykaryocytes became visible in rats receiving different types of fibres, no further difference could be recognized in either morphology or mode of polykaryocyte production in the experimental groups. The finding that fibrous quartz and glass fibres induced giant cell formation whereas DQ 12 and powdered glass did not, suggests that the fibrous shape of the dusts rather than their mineral or chemical composition determines their biological effects. This confirms previous interpretations stressing the importance of the physical properties of fibres for tumour induction. (BECK and BRUCH, 1974; POTT and FRIEDRICHS, 1972; STANTON and WRENCH, 1972). The results of the present study appear to confirm some of the previous suggestions on the mode of formation of polykaryocytes. Contiguous cells are often seen elaborating ectoplasmic interdigitations and it has been suggested this may lead to cell fusion by periodic breaks in apposed membranes (POLICARD et al., 1965). Occurrence of cytoplasmic contacts between macrophages has long been recognized, and ARONSON (1963) has demonstrated transmission of killed tubercle bacilli and ribonucleic acid molecules via intercytoplasmic bridges. Diffusion chamber studies give evidence that macrophages can form syncytia (ESKELAND and KJAERHEIM, 1966), and lysosomes have been said to play an active part in the process of cell fusion (SUTTON, 1966). POSTE and REEVE (1972) and RUBIN (1968) have suggested that the removal of Ca 2 + ions from the cellular membrane leads to depolarized areas which can subsequently fuse with corresponding areas of neighbouring cells. Furthermore, it has been suggested that both anaesthetics and tranquillizers can occupy the Ca 2 + free areas and thus make them unavailable for fusion. In the present study, the morphological pattern and lysosomal changes observed in our cells closely correspond to those described by POLICARD et al. (1965) and SUTTON (1966). We have also observed a secondary incorporation of normal cells in experimentally-induced polykaryocytes. This induction of polykaryocytes was found to be prevented by treatment with anaesthetic and tranquillizer. This group of results not only confirms the suggestion that polykaryocytes are produced by a process of cell fusion, but also supports the idea that this fusion is initiated by the removal of Ca 2 + ions from areas of the cell membrane. However, it must be emphasized that the exact site of drug action is not clear. Also it is difficult to distinguish between the effects of drugs on the fluxes into or from the membrane-bound pool of Ca 2 + and the transmembrane fluxes of Ca 2 + into or from cytoplasm (CURTIS, 1970; KALIX, 1971). However, Ca 2 + concentration in the
Induction of polykaryocytes
177
membrane phase is 10 to 20 times higher than in the cytoplasm, and this fact must be taken into consideration while interpreting the results (CHU-WONQ and SEEMANN, 1971). REFERENCES ARONSON, M. (1963) J. exp. Med. 118, 1083.
POLICARD, A., COLLET, A., MARTIN, J. C. and RENT, C. (1965) Z. Zellforsch. Mikrosk. Anat. 66,
96-105. POSTE, G. and REEVE, P. (1972) Expl. Cell Res. 72, 556-60. POTT, F. and FRIEDRICHS, K.-H. (1972) Natunvissenschafien, 59, 318.
ROBOCK, K. (1973) Ann. occup. Hyg. 16, 63-66. RUBIN, R. P. (1968) Pharmac. Rev. 22, 389^28. SETHI, S., BECK, E. G. and MANOJLOVIC, N. (1974) Ann. occup. Hyg. 17, 53-56.
STANTON, M. F. and WRENCH, C. (1972) / . natn Cancer Inst. 48, 797-821. SUTTON, J. S. (1966) Natn. Cancer Inst. Monogr. 26, 71-144.
Downloaded from http://annhyg.oxfordjournals.org/ at The University of British Colombia Library on June 21, 2015
BECK, E. G. and BRUCH, J. (1974) Rev. franc. Mai. Resp. 1, 72-76. CHU-WONG, M. and SEEMANN, P. (1971) Btochim. Biophys. Ada 241, 473-82. CURTIS, B. A. (1970) / . gen. Physiol. 55, 243-53. ESKELAND, G. and KJAERHEIM, A. (1966) Ada path, microbiol. scand. 68, 549. KAUX, A. (1971) Pftiigers Arch. ges. Physiol. 326, 1-14.
THE INDUCTION OF POLYKARYOCYTES BY VARIOUS FIBROUS DUSTS AND THEIR INHIBITION BY DRUGS IN RATS N. MANOJLOVIC*
Abstract—U.I.C.C. chrysotile, glass fibres, and fibrous quartz were intraperitoneally administered to rats using the cover slip method. The animals were sacrificed at 24-hr intervals and the cover slips prepared for phase contrast and interference microscopy. On the first days mononuclear dendritic and epitheloid macrophages were observed, which had phagocytosed fibrous material and deposited it perinuclearly in lysosomes. Between the fourth and fifth day polykaryocytes appeared, which showed variable sizes and secondary integration of mononuclear cells. Giant cell morphology and mode of development were independent of the type of fibre administered. Control animals which had received Docrentrupper quartz, powdered glass or physiological saline, showed only mononuclear cellular reaction during eight days of the experiment. Animals which had received U.I.C.C. crocidolite were treated with meprobamate and procaine hydrochloride on the third to the fifth day. In this case no polykaryocytes were found. The mechanism of polykaryocyte induction and the influence of the drugs as regards inhibition of polykaryocyte development are discussed.
INTRODUCTION
IN AN earlier report we have described the development and morphology of polykaryocytes following intraperitoneal administration of U.I.C.C. crocidolite to rats (SETHI et al., 1974). In the present investigation, the morphology of polykaryocytes induced by U.I.C.C. chrysotile, glass fibres, and fibrous quartz have been compared under similar experimental conditions. In addition, the influence of intraperitoneallyadministered procaine and meprobamate on the development of polykaryocytes induced by crocidolite has been studied. MATERIALS As experimental dusts U.I.C.C. chrysotile, glass fibre, fibrous quartz and, for the drug effect, U.I.C.C. crocidolite were employed. The glass fibre and fibrous quartz samples ranged in length from 1 to 20 /im and in diameter from 0-25 to 1 fim. Substances used for control groups were Doerentruper quartz (DQ 12, ROBOCK, 1973) powdered glass, and pyrogen-free physiological saline. Both DQ 12 and the powdered glass had particle sizes < 3 /im. Procaine hydrochloride and meprobamate (2-methyl-2-n-propyl-l,3-propanedioldicarbamate) were prepared as TyTode's solutions in a concentration of 10 mg/ml. • Med. Institut fur Lufthygjene und Silikoseforschung an der Universitat Dusseldorf. t Institut fur Hygiene im Zentrum fttr Okologie der Justus-Liebig-Universitat Giessen (BRD). Please address requests for reprints to Prof. Beck, Hygiene-Institut der Universitat, 6300 Giessen, Friedrichstr. 16, BRD. 173
Downloaded from http://annhyg.oxfordjournals.org/ at The University of British Colombia Library on June 21, 2015
S. SETHI,* E. G. BECK! and
174
S. SETHI, E. G. BECK and N. MANOJLOVIC
METHODS
Procaine (LD 30 = 25 mg/100 g body wt. rat) 6 rats: five 5 mg doses per day, on days 3, 4 and 5 4 rats: five 5 mg doses per day, on days 4 and 5 6 rats: five 7 -5 mg doses per day on days 3, 4 and 5 4 rats: five 7-5 mg doses per day, on days 4 and 5. Meprobamate (LD J 0 = 41 mg/100 g body wt. rat) 6 rats: five 6 mg doses per day, on days 3, 4 and 5 4 rats: five 6 mg doses per day, on days 4 and 5 6 rats: five 12 mg doses per day, on days 3, 4 and 5 4 rats: five 12 mg doses per day, on days 4 and 5. All animals were killed on the sixth day and cover slips were taken out and processed for phase and interference contrast microscopy. RESULTS Groups treated with fibres only The cellular reaction observed during the first three days following administration of three different fibre types was identical. One side of the cover slips was covered with omental tissue and large deposits of the substance administered, and the other side was covered with a monolayer of mononucleated cells mainly consisting of epitheloid and dendritic macrophages. Mostly the dust was lying within lysosomes which were perinuclearly arranged and only a few fibres were seen lying free in the cytoplasm (Fig. 1). After administration of fibrous quartz, the cells often showed necrobiotic alterations of variable degree, particularly those which were heavily packed with fibres. The cellular reaction after the third day following the administration of dusts differed in the various animal groups: In the animals which had received glass fibres or chrysotile, polykaryocytes became visible on the fourth day. Many round and oval nuclei with prominent nucleoli were lying in groups within the cell. The cytoplasm was mostly homogeneous and lacked structures except for the occasional presence of glass fibres and the vacuolation in the neighbourhood of the nuclei. The cellular membrane appeared thickened due to its infolding and high membranous activity.
Downloaded from http://annhyg.oxfordjournals.org/ at The University of British Colombia Library on June 21, 2015
A total of 136 female Wistar rats was employed. Three groups of 24 animals each were given U.I.C.C. chrysotile A, glass fibres, and fibrous quartz respectively. Three rats of each group were sacrificed at intervals of 24 hr from the first to the 8th day. Three control groups of 8 rats each received glass powder, DQ 12 quartz and physiological saline (2 ml/rat) respectively. An animal of each group was sacrificed at intervals of 24 hr from the first to the 8th day. All dusts were administered peritoneally in doses of 5mg/rat, using the cover slip method. After sacrificing the animals, implanted cover slips were taken out and prepared for microscopical examination as described by SETHI et al (1974). Forty rats received crocidolite on day 1 and were then divided into two groups and treated with procaine or meprobamate respectively by peritoneal injection as follows:
(facing page 174)
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FIG. 1. 48 hr. Dendritic and epitheloid macrophages. Arrows indicate glass fibres lying in lysosomes arranged perinuclearly, with a few fibres lying free in the cytoplasm. (a) Phase contrast 1 240 x . (b) Interference contrast 1 240 x . FIG. 2. 5 days. Polykaryocytes induced by glass fibres with variable cytoplasmic and nuclear morphology, (a) Groups of nuclei (thick arrow) are surrounded by numerous vacuoles (thin arrow) Homogeneous, structureless cytoplasm contains engulfed fibrous material (short arrow), (b) Groups of darker and lighter nuclei are randomly distributed in the cell (arrows). Phase contrast 1 240 x .
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FIG. 3. 5 days. Secondary incorporation of mononuclear cells by polykaryocytes induced by glass fibres, (a) Several incorporated cells lie in vacuoles within a polykaryocyte (arrows). These cells surround another incorporated cell which is merging with the cytoplasm of the giant cell. Nearby a nucleus is free in the cytoplasm with vacuoles. (b) Incorporated cells in a vacuole within a polykaryocyte and already integrated nuclei (arrows). Phase contrast 1 240 x . FIG. 4. 5 days. Strands of nuclei (arrows) stretching from the periphery to the deeper regions of a polykaryocyte induced by glass fibres. These nuclei are probably derived from secondarily integrated cells, (a) Interference contrast 530 x . (b) Phase contrast 1 240 x. FIG. 5. 6 days. No development of polykaryocytes after simultaneous treatment with crocidolite and meprobamate. Only dendritic and epitheloid macrophages are seen. Phase contrast 1 240 x .
Induction of polykaryocytes
175
Control groups In the groups treated with DQ 12 or powdered glass the early cellular reaction from the first to the third day was similar to that seen in the experimental groups. The mononuclear dendritic and epitheloid macrophages were heavily packed with administered dust. After saline administration the cells were highly vacuolated. Epitheloid cells were rare compared with the experimental groups. Mononuclear cells persisted until the end of the experiment. The only alteration observed was the enlargement of the lysosomes—the typical cytotoxic effect of DQ 12. Meprobamate and procaine groups On the sixth day, all the animals regardless of dose type and duration of drug administration showed a similar cellular reaction.
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On the fifth day, the polykaryocytes formed large syncytia, showing variable cytoplasmic and nuclear morphology at different regions. Groups of darker nuclei lying in the vicinity of darker areas of plasma and groups of lighter nuclei were randomly distributed in the cell. The cytoplasm became vacuolated, homogeneous without showing structures, or contained fibrous material (Fig. 2). The presence of single undamaged cells within the polykaryocytes was striking. These cells seemed to be engulfed secondarily. A mononuclear cell was first seen lying within a concavity formed by the cytoplasmic pseudopodia, being gradually taken up by the polykaryocytes and then lying inside a vacuole within the polykaryocyte (Fig. 3). Starting from one end, the cytoplasm of the incorporated cell finally merged with the main plasm. This process continued until the integrated cell lost its identity and only the nucelus could be made out. Sometimes strands of nuclei stretching from the periphery to the deeper regions were observed. These nuclei probably belonged to the secondarily incorporated cells being pushed deeper into the polykaryocyte as numerous cells were incorporated from the periphery (Fig. 4). All these characteristic symptoms developed until the end of the 8th day of the experiment. Except for the necrobiotic alteration in the cells, development and appearance of polykaryocytes were essentially the same in the animals receiving fibrous quartz as in those receiving glass fibre. Polykaryocytes containing a few nuclei at first appeared on the fourth day and showed a continuous increase in size and number of nuclei until the 8th day. Cellular morphology was also the same as that seen in animals after glass fibre application. The proximity of mononuclear and multinuclear cells and their cytoplasmic extensions was very striking, as well as the demonstration of morphologically-different areas within a single polykaryocyte. This process represents polykaryocyte formation being mediated by the process of fusion. Polykaryocytes became visible on the sixth day following administration of chrysotile fibres. Polykaryocytes at this stage contained only a few nuclei arranged in rosette form within a highly-vacuolated cytoplasm. On the 7th and 8th day, large polykaryocytes containing large numbers of nuclei (often more than 50) were observed. Other cytoplasmic and nuclear characteristics were similar to those in polykaryocytes induced by glass fibres. An important feature of these polykaryocytes was the fact that while most cells joining the main syncytial mass were mononuclear, some already contained two or three nuclei.
176
S. S m n ,
E. G. BECK and N. MANOJLOVIC
The cell population consisted of mononuclear dendritic and epitheloid macrophages which contained phagocytosed crocidolite asbestos fibres lying within lysosomes. Most of the cells were highly vacuolated. Occasionally some cells were rounded up and these showed no vacuolation. No polykaryocytes were observed in any group, regardless of dose and duration of administration of meprobamate and procaine hydrochloride (Fig. 5).
Downloaded from http://annhyg.oxfordjournals.org/ at The University of British Colombia Library on June 21, 2015
DISCUSSION This method, which involves implantation of glass cover slips into the rat peritoneal cavity along with experimental dusts, followed by their removal at indicated intervals, allows constant in vitro observations of events induced in vivo (SETHI et al., 1974). Except for the different time intervals at which polykaryocytes became visible in rats receiving different types of fibres, no further difference could be recognized in either morphology or mode of polykaryocyte production in the experimental groups. The finding that fibrous quartz and glass fibres induced giant cell formation whereas DQ 12 and powdered glass did not, suggests that the fibrous shape of the dusts rather than their mineral or chemical composition determines their biological effects. This confirms previous interpretations stressing the importance of the physical properties of fibres for tumour induction. (BECK and BRUCH, 1974; POTT and FRIEDRICHS, 1972; STANTON and WRENCH, 1972). The results of the present study appear to confirm some of the previous suggestions on the mode of formation of polykaryocytes. Contiguous cells are often seen elaborating ectoplasmic interdigitations and it has been suggested this may lead to cell fusion by periodic breaks in apposed membranes (POLICARD et al., 1965). Occurrence of cytoplasmic contacts between macrophages has long been recognized, and ARONSON (1963) has demonstrated transmission of killed tubercle bacilli and ribonucleic acid molecules via intercytoplasmic bridges. Diffusion chamber studies give evidence that macrophages can form syncytia (ESKELAND and KJAERHEIM, 1966), and lysosomes have been said to play an active part in the process of cell fusion (SUTTON, 1966). POSTE and REEVE (1972) and RUBIN (1968) have suggested that the removal of Ca 2 + ions from the cellular membrane leads to depolarized areas which can subsequently fuse with corresponding areas of neighbouring cells. Furthermore, it has been suggested that both anaesthetics and tranquillizers can occupy the Ca 2 + free areas and thus make them unavailable for fusion. In the present study, the morphological pattern and lysosomal changes observed in our cells closely correspond to those described by POLICARD et al. (1965) and SUTTON (1966). We have also observed a secondary incorporation of normal cells in experimentally-induced polykaryocytes. This induction of polykaryocytes was found to be prevented by treatment with anaesthetic and tranquillizer. This group of results not only confirms the suggestion that polykaryocytes are produced by a process of cell fusion, but also supports the idea that this fusion is initiated by the removal of Ca 2 + ions from areas of the cell membrane. However, it must be emphasized that the exact site of drug action is not clear. Also it is difficult to distinguish between the effects of drugs on the fluxes into or from the membrane-bound pool of Ca 2 + and the transmembrane fluxes of Ca 2 + into or from cytoplasm (CURTIS, 1970; KALIX, 1971). However, Ca 2 + concentration in the
Induction of polykaryocytes
177
membrane phase is 10 to 20 times higher than in the cytoplasm, and this fact must be taken into consideration while interpreting the results (CHU-WONQ and SEEMANN, 1971). REFERENCES ARONSON, M. (1963) J. exp. Med. 118, 1083.
POLICARD, A., COLLET, A., MARTIN, J. C. and RENT, C. (1965) Z. Zellforsch. Mikrosk. Anat. 66,
96-105. POSTE, G. and REEVE, P. (1972) Expl. Cell Res. 72, 556-60. POTT, F. and FRIEDRICHS, K.-H. (1972) Natunvissenschafien, 59, 318.
ROBOCK, K. (1973) Ann. occup. Hyg. 16, 63-66. RUBIN, R. P. (1968) Pharmac. Rev. 22, 389^28. SETHI, S., BECK, E. G. and MANOJLOVIC, N. (1974) Ann. occup. Hyg. 17, 53-56.
STANTON, M. F. and WRENCH, C. (1972) / . natn Cancer Inst. 48, 797-821. SUTTON, J. S. (1966) Natn. Cancer Inst. Monogr. 26, 71-144.
Downloaded from http://annhyg.oxfordjournals.org/ at The University of British Colombia Library on June 21, 2015
BECK, E. G. and BRUCH, J. (1974) Rev. franc. Mai. Resp. 1, 72-76. CHU-WONG, M. and SEEMANN, P. (1971) Btochim. Biophys. Ada 241, 473-82. CURTIS, B. A. (1970) / . gen. Physiol. 55, 243-53. ESKELAND, G. and KJAERHEIM, A. (1966) Ada path, microbiol. scand. 68, 549. KAUX, A. (1971) Pftiigers Arch. ges. Physiol. 326, 1-14.
