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Reversible inhibition of DNA, RNA and protein synthesis in human cells by lead chloride
Toxicology, 5 (1975) 167--174 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
R E V E R S I B L E INHIBITION OF DNA, R N A AND P R O T E I N SYNTHESIS IN HUMAN CELLS BY LEAD CHLORIDE
YVETTE ~KREB and VLASTA HABAZIN-NOVAK
Institute for Medical Research and Occupational Health, Zagreb (Yugoslavia) (Received April 22nd, 1975) (Accepted July ]0th, 1975)
SUMMARY
An acute intoxication by lead chloride (conc. 2.5" 1 0 - 4 M ) produces a temporary reduction of macromolecular syntheses in HeLa cells growing asynchronously. The reduction is similar for DNA, RNA and proteins and differs only in the intensity. After a one-day intoxication, if the cells are put back in a fresh medium, the syntheses return to normal within 10 h. The histochemical sulphide-silver m e t h o d shows that lead is present in the cells during the inhibition.
INTRODUCTION
Cellular response to lead has several characteristic features [1]. The best known are the formation of intranuclear inclusion bodies, mitochondrial alterations, modifications of protein synthesis and cytogenetic effects. G o y e r reported that it would be useful to better understand the metabolism of lead particularly in terms of its effects on cellular organelles and cell metabolism [ 2]. Our intention was to pay particular attention to the effects which precede evident toxic manifestations. It has been reported that lead apparently acts at a large number of biochemical sites and that it might contribute to metabolic alterations through several different mechanisms [3]. Among them we can mention the effects on nucleic acids and protein metabolism. It has been suggested that modifications in the nucleic acid content of leucocytes in patients intoxicated by lead can serve to assess the severity of lead poisoning [4]. Later it was demonstrated that lead interacts with ribonucleic acid in the liver of lead-poisoned ponies [3]. Enzymatic activity is also strongly modified in tissues and blood of lead-poisoned humans and animals [5]. It Abbreviations: PBS, phosphate-buffered saline; TCA, trichloroacetic acid.
167
therefore seems to us that alterations in the DNA, RNA and protein synthesis m a y be a sensitive enough parameter to evaluate the primary effects of lead on the cells. In spite of the fact that the results of experiments in vitro are not always comparable with those in vivo, we think that human cells in tissue culture represent a convenient system for these studies. Although the relevant literature is very poor, HeLa cells appear to be an appropriate material. They were already successfully used for similar investigations of the effects of various toxic agents on cell metabolism [6,7]. The present paper is an experimental study of the response of DNA, R N A and protein synthesis to an acute intoxication by lead chloride in HeLa cells growing asynchronously. MATERIAL AND METHODS
Cell culture The cells were a sub-line derived from HeLa cells used in the Rudjer Bo~kovid Institute, Zagreb. They were grown as a monolayer and were routinely passaged in MEM Eagle's medium supplemented with 10% calf serum and antibiotics. Two days after subcultivation the cells were trypsinized, counted with a h a e m o c y t o m e t e r and atiquots of 105 cells transferred with 1 ml medium into sterile vials used for scintillation counting. 40 h later the cells were ready for experimental purpose. Acute intoxication Lead chloride (p.a. Kemika, Zagreb) was added to the medium in half of the vials, at a concentration of 2 . 5 . 1 0 - 4 M (69.5 pg/ml). This is the highest concentration which did not change the pH of the medium and did n o t induce any precipitate. The incubation time varied from 1 to 30 h. Biological parameter To assay the first biological effe.ct of lead the number of cells was frequently recorded enabling us to evaluate the growth of asynchronous populations of control and treated cells. Continuous labelling o f the cells The cells incubated with PbC12 as well as the control cells were submitted to the same procedure. For labelling studies we adapted the method of Miller et al. [ 8 ] . For DNA synthesis [ 3 H ] t h y m i d i n e was added to each vial (conc. 0.2 gCi/ml; spec. act. 5 Ci/mmole; Amersham, UK). The R N A synthesis was followed b y the uptake of [SH] uridine (conc. 0.2 pCi/ml; spec. act. 1.52 Ci/mmole; Amersham, UK). To control the incorporation of [SH]uridine into RNA, a test with deoxyribonuclease (Sigma, USA) was also done. The protein synthesis was followed by the uptake of [14C]phenylalanine, conc. 0.2 pCi/ml; spec. act. 6 mCi/mmole; Amersham, UK). This precursor was added at the same time as [s H] uridine. 168
At regular intervals from 1 to 30 h the radioactive medium was removed from 6 vials (3 controls and 3 with lead-poisoned cells). The cells were carefully rinsed six times, with PBS solution, rapidly fixed by cold Carnoy's fluid, rinsed three times with cold 5% TCA, and dried. 5 ml of scintillation liquid (4 g PPO and 50 mg POPOP in 1 liter toluene) was added in each vial. The radioactivity of the samples was counted in a Packard liquid scintillation spectrophotometer.
Pulse labelling of the cells after a 24-h pretreatment with lead chloride The cells were incubated with PbC12 during 24 h as described previously. The contaminated medium was removed, the cells were carefully rinsed 6 times and incubated in a fresh medium with radioactive thymidine, uridine or phenylalanine. The incorporation of precursors was followed by a 30-min pulse-labelling every 2 h during 10 h. The [3H] thymidine, [3HI uridine and [14C] phenylalanine uptake was evaluated in the same way as in the case of continuous labelling. Recording of the results The a m o u n t of radioactivity of the cells recorded as cpm" 1 0 - 3 / p e r sample was expressed as a function of time in hours. On the graphs each symbol represents the means of 3 measurements from 3 parallel samples. The standard errors were not reported because they were insignificant. Each experiment was repeated at least three times. For the sake of clarity only the results of a typical experiment are shown in each group. Histochemical sulphide-silver method To detect if lead is really taken up by the cells we performed an adaptation of the sulphide-silver m e t h o d of Brunk and Brun [9]. This m e t h o d is based on the principle that heavy metals in tissues when transformed into sulphide may catalyse the reduction of Ag 2+ into Ag. After various incubation times with lead chloride, the cells were fixed for 2 min in 96% cold ethanol, directly exposed for 1 h to a 2% solution of ammonium sulphide in 70% ethanol, rinsed in 96% ethanol and in glassdistilled water and developed for 2.5 h in a fresh mixture of 20% gum Arabic (100 ml), 10% AgNO3 (1 ml), 5 g citric acid and 2 g hydroquinone in 100 ml (10 ml). After careful rinsing, the cells were exposed to 5% TCA for 10 min to eliminate the traces of the other possible contaminant metals. In the cells in which black granules appeared the lead is supposed to have been present. Simultaneously, control groups were submitted to the same procedure. RESULTS
Biological effect In our experimental conditions, the average generation time of HeLa 169
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Fig. 1. E f f e c t o f lead c h l o r i d e on the c o n t i n u o u s labelling o f H e L a cells. Abscissa: t i m e o f i n c u b a t i o n w i t h PbCl2; ordinate" c p m • 1 0 - - 3 / s a m p l e o A [] c o n t r o l u n t r e a t e d cells"
• A, .., cells treated w i t h PbC] 2. ( l a ) [ H ] T h y m i d i n e uptake; (lb~ [ 3 H ] uridine uptake; ( l c ) [ 14 C ] phenylalanine uptake.
cells was a b o u t 20 h. During the acute intoxication after 34 h a prolongation of the generation time could be observed which finally reached 55 h after a 72-h incubation in lead. After that time, the cells began to lyse rapidly. During our experimental period the number of cells in the control and treated groups remained the same.
Long-term incorporation The results of one typical experiment are summarized graphically. The two curves in Fig. l a show the differences between the long-term incorporation of [3H] thymidine in the DNA of normal cells and the uptake of the precursor b y a population of cells incubated with lead chloride from 1 until 30 h. During the first 6 h the differences between the two groups are very small (10%) b u t after this lapse of time the differences increase sharply and the residual incorporation in the treated cells becomes about 40% of the controls. Figs. l b and l c show very similar results for the incorporation of [3H]uridine and [14C]phenylalanine into R N A and protein respectively. At the end of the experiment, the residual incorporation is approximately 37% of the control for R N A and only 27% for the protein synthesis.
Pulse labelling of the cells To follow what happens after this progressive inhibition a second t y p e of experiment was done.
171
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Fig. 2. E f f e c t o f a 24-h p r e t r e a t m e n t with lead chloride on the pulse-labelling o f H e L a cells. T h e l e g e n d is the s a m e as to Fig. 1. ( 2 a ) [ 3 H ] T h y m i d i n e u p t a k e ; (2b) [3H]uridine uptake;(2c)[14C]phenylalanine u p t a k e .
The cells were intoxicated during 24 h as before. The contaminated medium was removed and replaced by a fresh one containing the radioactive precursors. Their incorporation in DNA, RNA and proteins was followed by pulse-labelling technique during 30 min every 2 h. In Figs. 2a, 2b and 2c, the curves start with the initial differences observed previously. This means t h a t the residual incorporation began as 40% of the control for DNA synthesis, 37% for RNA and 27% for the proteins. Without lead the syntheses increased very quickly and 10 h later they were quite normal and did not differ from the controls.
Localisation o f lead To investigate whether there is a correlation between the inhibition of syntheses and the presence of lead in the cells we adapted the histochemical m e t h o d of Brunk et al. [9] as described in Materials and methods. It takes half an hour to see the first black lead granules in the cytoplasm of the cells. During the next few hours, the particles become larger, more numerous and preferentially accumulate around the nucleus. In some cases they are visible even inside the nucleus, but all the cells are not equally marked. After 6 h the number of granules has n o t increased; the granules only become larger and more confluent. If the cells are returned to the normal medium, the granules slowly disappear and after 24 h only a few of them are visible. This histochemical technique gives quite a good indication that lead is present in the cells as shown for fibroblasts [9]. The technique, however, should be considered more qualitative than quantitative. We can conclude that the distribution of the particles into the cells differs with the duration of incubation with lead and this particular distribution is worth commenting upon. DISCUSSION
The lead added to the medium is responsible for the macromolecular synthesis inhibition. All syntheses are affected in a similar manner. They only differ in intensity of inhibition. This agrees well with some other authors' findings who accord particular attention to the lead effect on the enzymatic system [ 5]. It therefore seems logical t h a t [ 14 C] phenylalanine uptake into the cells is the most affected. If lead enters quickly into HeLa cells it must reach a certain concentration to produce a measurable inhibition. It needs also a certain time for the elimination at a level which allows a return of the syntheses to the normal. In this first stage of our work, it was n o t y e t possible to distinguish if the inhibitory effect is due to the well-known binding of lead to various cytoplasmic and nuclear sites or if it is a consequence of an effect on the cellular membrane which can also partially prevent the entry of the radioprecursors. These two factors and some others are probably involved in the
173
inhibition which shows a certain similarity with that observed after the action of ethanol on HeLa cells in culture [10]. The data presented here are not sufficient to make a conclusion about a specific mechanism of the action of lead in terms of interference with the radioprecursor utilisation by human cells. However, the p h e n o m e n o n is sufficiently significant to warrant special attention to this simple cell culture model system for evaluation of the toxicity of heavy metals. The first results should stimulate further investigations in this field. ACKNOWLEDGEMENTS
The authors wish to thank Mrs. Nada Hor~ and Jadranka Ra~id for their skillful technical assistance and Dr. Ulf Brunk for his valuable advice. This work was supported by a grant from the Council for Scientific Work of Croatia. REFERENCES 1 R.A. Goyer and B.C. Rhyne, Int. Rev. Exptl. Pathol., 12 (1973) 1. 2 R.A. Goyer, Arch. Environ. Health, 20 (1970) 705. 3 D.D. Ulmer and B.L. Vallee, in A.D. Hemphill (Ed.), Trace Substances in Environmental Health, If, Univ. of Missouri, 1968, p. 7. 4 C.A. Nunziante and A. Granata, Arch. Mal. Prof., 18 (1957) 412. 5 B.L. Vallee and D.D. Ulmer, Ann. Rev. Biochem., 41 (1972) 92. 6 C.L. Litterstand E.P. Lichtenstein,Arch. Environ. Health, 22 (1971) 454. 7 L. Spangberg and K. Langeland, Oral Surg., 35 (1973) 402. 8 G.G. Miller,G.W.R. Walker and R.E. Giblak, Exptl. Cell.Res., 72 (1972) 533. 9 U. Brunk and A. Brun, Histochemie, 29 (1972) 140. 10 F. Koch and G. Koch, Res. C o m m u n . Chem. Pathol. Pharmacol., 9 (1974) 291.
174
R E V E R S I B L E INHIBITION OF DNA, R N A AND P R O T E I N SYNTHESIS IN HUMAN CELLS BY LEAD CHLORIDE
YVETTE ~KREB and VLASTA HABAZIN-NOVAK
Institute for Medical Research and Occupational Health, Zagreb (Yugoslavia) (Received April 22nd, 1975) (Accepted July ]0th, 1975)
SUMMARY
An acute intoxication by lead chloride (conc. 2.5" 1 0 - 4 M ) produces a temporary reduction of macromolecular syntheses in HeLa cells growing asynchronously. The reduction is similar for DNA, RNA and proteins and differs only in the intensity. After a one-day intoxication, if the cells are put back in a fresh medium, the syntheses return to normal within 10 h. The histochemical sulphide-silver m e t h o d shows that lead is present in the cells during the inhibition.
INTRODUCTION
Cellular response to lead has several characteristic features [1]. The best known are the formation of intranuclear inclusion bodies, mitochondrial alterations, modifications of protein synthesis and cytogenetic effects. G o y e r reported that it would be useful to better understand the metabolism of lead particularly in terms of its effects on cellular organelles and cell metabolism [ 2]. Our intention was to pay particular attention to the effects which precede evident toxic manifestations. It has been reported that lead apparently acts at a large number of biochemical sites and that it might contribute to metabolic alterations through several different mechanisms [3]. Among them we can mention the effects on nucleic acids and protein metabolism. It has been suggested that modifications in the nucleic acid content of leucocytes in patients intoxicated by lead can serve to assess the severity of lead poisoning [4]. Later it was demonstrated that lead interacts with ribonucleic acid in the liver of lead-poisoned ponies [3]. Enzymatic activity is also strongly modified in tissues and blood of lead-poisoned humans and animals [5]. It Abbreviations: PBS, phosphate-buffered saline; TCA, trichloroacetic acid.
167
therefore seems to us that alterations in the DNA, RNA and protein synthesis m a y be a sensitive enough parameter to evaluate the primary effects of lead on the cells. In spite of the fact that the results of experiments in vitro are not always comparable with those in vivo, we think that human cells in tissue culture represent a convenient system for these studies. Although the relevant literature is very poor, HeLa cells appear to be an appropriate material. They were already successfully used for similar investigations of the effects of various toxic agents on cell metabolism [6,7]. The present paper is an experimental study of the response of DNA, R N A and protein synthesis to an acute intoxication by lead chloride in HeLa cells growing asynchronously. MATERIAL AND METHODS
Cell culture The cells were a sub-line derived from HeLa cells used in the Rudjer Bo~kovid Institute, Zagreb. They were grown as a monolayer and were routinely passaged in MEM Eagle's medium supplemented with 10% calf serum and antibiotics. Two days after subcultivation the cells were trypsinized, counted with a h a e m o c y t o m e t e r and atiquots of 105 cells transferred with 1 ml medium into sterile vials used for scintillation counting. 40 h later the cells were ready for experimental purpose. Acute intoxication Lead chloride (p.a. Kemika, Zagreb) was added to the medium in half of the vials, at a concentration of 2 . 5 . 1 0 - 4 M (69.5 pg/ml). This is the highest concentration which did not change the pH of the medium and did n o t induce any precipitate. The incubation time varied from 1 to 30 h. Biological parameter To assay the first biological effe.ct of lead the number of cells was frequently recorded enabling us to evaluate the growth of asynchronous populations of control and treated cells. Continuous labelling o f the cells The cells incubated with PbC12 as well as the control cells were submitted to the same procedure. For labelling studies we adapted the method of Miller et al. [ 8 ] . For DNA synthesis [ 3 H ] t h y m i d i n e was added to each vial (conc. 0.2 gCi/ml; spec. act. 5 Ci/mmole; Amersham, UK). The R N A synthesis was followed b y the uptake of [SH] uridine (conc. 0.2 pCi/ml; spec. act. 1.52 Ci/mmole; Amersham, UK). To control the incorporation of [SH]uridine into RNA, a test with deoxyribonuclease (Sigma, USA) was also done. The protein synthesis was followed by the uptake of [14C]phenylalanine, conc. 0.2 pCi/ml; spec. act. 6 mCi/mmole; Amersham, UK). This precursor was added at the same time as [s H] uridine. 168
At regular intervals from 1 to 30 h the radioactive medium was removed from 6 vials (3 controls and 3 with lead-poisoned cells). The cells were carefully rinsed six times, with PBS solution, rapidly fixed by cold Carnoy's fluid, rinsed three times with cold 5% TCA, and dried. 5 ml of scintillation liquid (4 g PPO and 50 mg POPOP in 1 liter toluene) was added in each vial. The radioactivity of the samples was counted in a Packard liquid scintillation spectrophotometer.
Pulse labelling of the cells after a 24-h pretreatment with lead chloride The cells were incubated with PbC12 during 24 h as described previously. The contaminated medium was removed, the cells were carefully rinsed 6 times and incubated in a fresh medium with radioactive thymidine, uridine or phenylalanine. The incorporation of precursors was followed by a 30-min pulse-labelling every 2 h during 10 h. The [3H] thymidine, [3HI uridine and [14C] phenylalanine uptake was evaluated in the same way as in the case of continuous labelling. Recording of the results The a m o u n t of radioactivity of the cells recorded as cpm" 1 0 - 3 / p e r sample was expressed as a function of time in hours. On the graphs each symbol represents the means of 3 measurements from 3 parallel samples. The standard errors were not reported because they were insignificant. Each experiment was repeated at least three times. For the sake of clarity only the results of a typical experiment are shown in each group. Histochemical sulphide-silver method To detect if lead is really taken up by the cells we performed an adaptation of the sulphide-silver m e t h o d of Brunk and Brun [9]. This m e t h o d is based on the principle that heavy metals in tissues when transformed into sulphide may catalyse the reduction of Ag 2+ into Ag. After various incubation times with lead chloride, the cells were fixed for 2 min in 96% cold ethanol, directly exposed for 1 h to a 2% solution of ammonium sulphide in 70% ethanol, rinsed in 96% ethanol and in glassdistilled water and developed for 2.5 h in a fresh mixture of 20% gum Arabic (100 ml), 10% AgNO3 (1 ml), 5 g citric acid and 2 g hydroquinone in 100 ml (10 ml). After careful rinsing, the cells were exposed to 5% TCA for 10 min to eliminate the traces of the other possible contaminant metals. In the cells in which black granules appeared the lead is supposed to have been present. Simultaneously, control groups were submitted to the same procedure. RESULTS
Biological effect In our experimental conditions, the average generation time of HeLa 169
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Fig. 1. E f f e c t o f lead c h l o r i d e on the c o n t i n u o u s labelling o f H e L a cells. Abscissa: t i m e o f i n c u b a t i o n w i t h PbCl2; ordinate" c p m • 1 0 - - 3 / s a m p l e o A [] c o n t r o l u n t r e a t e d cells"
• A, .., cells treated w i t h PbC] 2. ( l a ) [ H ] T h y m i d i n e uptake; (lb~ [ 3 H ] uridine uptake; ( l c ) [ 14 C ] phenylalanine uptake.
cells was a b o u t 20 h. During the acute intoxication after 34 h a prolongation of the generation time could be observed which finally reached 55 h after a 72-h incubation in lead. After that time, the cells began to lyse rapidly. During our experimental period the number of cells in the control and treated groups remained the same.
Long-term incorporation The results of one typical experiment are summarized graphically. The two curves in Fig. l a show the differences between the long-term incorporation of [3H] thymidine in the DNA of normal cells and the uptake of the precursor b y a population of cells incubated with lead chloride from 1 until 30 h. During the first 6 h the differences between the two groups are very small (10%) b u t after this lapse of time the differences increase sharply and the residual incorporation in the treated cells becomes about 40% of the controls. Figs. l b and l c show very similar results for the incorporation of [3H]uridine and [14C]phenylalanine into R N A and protein respectively. At the end of the experiment, the residual incorporation is approximately 37% of the control for R N A and only 27% for the protein synthesis.
Pulse labelling of the cells To follow what happens after this progressive inhibition a second t y p e of experiment was done.
171
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Fig. 2. E f f e c t o f a 24-h p r e t r e a t m e n t with lead chloride on the pulse-labelling o f H e L a cells. T h e l e g e n d is the s a m e as to Fig. 1. ( 2 a ) [ 3 H ] T h y m i d i n e u p t a k e ; (2b) [3H]uridine uptake;(2c)[14C]phenylalanine u p t a k e .
The cells were intoxicated during 24 h as before. The contaminated medium was removed and replaced by a fresh one containing the radioactive precursors. Their incorporation in DNA, RNA and proteins was followed by pulse-labelling technique during 30 min every 2 h. In Figs. 2a, 2b and 2c, the curves start with the initial differences observed previously. This means t h a t the residual incorporation began as 40% of the control for DNA synthesis, 37% for RNA and 27% for the proteins. Without lead the syntheses increased very quickly and 10 h later they were quite normal and did not differ from the controls.
Localisation o f lead To investigate whether there is a correlation between the inhibition of syntheses and the presence of lead in the cells we adapted the histochemical m e t h o d of Brunk et al. [9] as described in Materials and methods. It takes half an hour to see the first black lead granules in the cytoplasm of the cells. During the next few hours, the particles become larger, more numerous and preferentially accumulate around the nucleus. In some cases they are visible even inside the nucleus, but all the cells are not equally marked. After 6 h the number of granules has n o t increased; the granules only become larger and more confluent. If the cells are returned to the normal medium, the granules slowly disappear and after 24 h only a few of them are visible. This histochemical technique gives quite a good indication that lead is present in the cells as shown for fibroblasts [9]. The technique, however, should be considered more qualitative than quantitative. We can conclude that the distribution of the particles into the cells differs with the duration of incubation with lead and this particular distribution is worth commenting upon. DISCUSSION
The lead added to the medium is responsible for the macromolecular synthesis inhibition. All syntheses are affected in a similar manner. They only differ in intensity of inhibition. This agrees well with some other authors' findings who accord particular attention to the lead effect on the enzymatic system [ 5]. It therefore seems logical t h a t [ 14 C] phenylalanine uptake into the cells is the most affected. If lead enters quickly into HeLa cells it must reach a certain concentration to produce a measurable inhibition. It needs also a certain time for the elimination at a level which allows a return of the syntheses to the normal. In this first stage of our work, it was n o t y e t possible to distinguish if the inhibitory effect is due to the well-known binding of lead to various cytoplasmic and nuclear sites or if it is a consequence of an effect on the cellular membrane which can also partially prevent the entry of the radioprecursors. These two factors and some others are probably involved in the
173
inhibition which shows a certain similarity with that observed after the action of ethanol on HeLa cells in culture [10]. The data presented here are not sufficient to make a conclusion about a specific mechanism of the action of lead in terms of interference with the radioprecursor utilisation by human cells. However, the p h e n o m e n o n is sufficiently significant to warrant special attention to this simple cell culture model system for evaluation of the toxicity of heavy metals. The first results should stimulate further investigations in this field. ACKNOWLEDGEMENTS
The authors wish to thank Mrs. Nada Hor~ and Jadranka Ra~id for their skillful technical assistance and Dr. Ulf Brunk for his valuable advice. This work was supported by a grant from the Council for Scientific Work of Croatia. REFERENCES 1 R.A. Goyer and B.C. Rhyne, Int. Rev. Exptl. Pathol., 12 (1973) 1. 2 R.A. Goyer, Arch. Environ. Health, 20 (1970) 705. 3 D.D. Ulmer and B.L. Vallee, in A.D. Hemphill (Ed.), Trace Substances in Environmental Health, If, Univ. of Missouri, 1968, p. 7. 4 C.A. Nunziante and A. Granata, Arch. Mal. Prof., 18 (1957) 412. 5 B.L. Vallee and D.D. Ulmer, Ann. Rev. Biochem., 41 (1972) 92. 6 C.L. Litterstand E.P. Lichtenstein,Arch. Environ. Health, 22 (1971) 454. 7 L. Spangberg and K. Langeland, Oral Surg., 35 (1973) 402. 8 G.G. Miller,G.W.R. Walker and R.E. Giblak, Exptl. Cell.Res., 72 (1972) 533. 9 U. Brunk and A. Brun, Histochemie, 29 (1972) 140. 10 F. Koch and G. Koch, Res. C o m m u n . Chem. Pathol. Pharmacol., 9 (1974) 291.
174