Cell Tiss. Res. 170, 289-303 (1976)
Cell and Tissue Research ~, by Springer-Verlag 1976
Mast Cell Activation and Tissue Cell Proliferation* Klas Norrby, Lennart EnerNick and Lennart Franz6n** *** Department of Pathology II, University of Link6ping, Sweden
Summary. The effect of mast cell activation and degranulation on the proliferation in the intact mesentery was studied in Sprague-Dawley rats. Mast cell activation was achieved by a single intraperitoneal injection of Compound 48/80. The proliferation was studied using three independent methods for estimation of cell production and D N A synthesis: 1. the mitotic index, 2. the relative number of cells having a D N A content in the S and G2 regions, by Feulgen photometric measurement in individual cells, and 3. the specific D N A activity, employing a method which combines a liquid scintillation technique after an intravenous injection of 3H-thymidine and Feulgen photometric determination of the D N A content per membrane preparation. It was found that the proliferation of the normal mesenchymal cells adjacent to the activated and degranulated mast cells in the mesentery was significantly increased within 24 and 32 h, the maximum increase being more than 20-fold compared to untreated controls. The results suggest that the c o m m o n type of mast cell may have a pathophysiological function related to stimulation of local cell proliferation.
Key words: Mast cell - C o m p o u n d 48/80 - Cell proliferation - Mesentery D N A - Mitosis.
Introduction A hypothetical relationship between mast cells and connective tissue activity has been discussed in the literature for a number of years (Riley, 1959; Selye, 1965; Send offprint requests to: Dr. Klas Norrby, Department of Pathology II, University of Link6ping, S 58185 Link6ping, Sweden
* Supported by grants from the Swedish Medical Research Council (Project 12X-2235) and from the Medical Faculty, Universityof Link6ping ** We thank Brita Saderlund, Margareta Oden6 and Irane Svensson for skilful technical assistance, and Erik Leander, Ph.D., for help with ~tatistical methods *** Part of this work was presented at the 7th Meeting of the European Study Group for Cell Proliferation, 5-9 May 1975, in Amsterdam, The Netherlands
290
K. Norrby et al.
F e r n e x , 1968). It was r e c e n t l y f o u n d in o u r l a b o r a t o r y ( N o r r b y , 1973b) t h a t the m a s t cell c o m p o n e n t s h e p a r i n , h i s t a m i n e a n d s e r o t o n i n at v e r y l o w c o n c e n t r a t i o n s , a l o n e o r m i x e d , release f i b r o b l a s t i c cells f r o m d e n s i t y - d e p e n d e n t i n h i b i t i o n o f cell d i v i s i o n . T h e s e f i n d i n g s in v i t r o s u g g e s t e d t h a t m a s t cell c o m p o n e n t s m i g h t also act as c e l l - d i v i s i o n - s t i m u l a t i n g a g e n t s in v i v o . T h e a i m o f the p r e s e n t w o r k was t o s t u d y the effect o f m a s t cell d e g r a n u l a t i o n o n the p r o l i f e r a t i o n o f a d j a c e n t n o r m a l m e s e n c h y m a l tissue cells. T h e s m a l l - g u t m e s e n t e r y o f t h e rat was c h o s e n as test tissue. M a s t cell d e g r a n u l a t i o n was a c h i e v e d by a single i n j e c t i o n o f C o m p o u n d 48/80. S u c h a t r e a t m e n t r e g u l a r l y i n d u c e d a m a r k e d p r o l i f e r a t i o n o f n e a r - b y f i b r o b l a s t i c a n d m e s o t h e l i a l cells. U s i n g a techn i q u e d e v e l o p e d in this l a b o r a t o r y ( E n e r b / i c k et al., 1976) the i n d u c e d p r o l i f e r a t i v e r e a c t i o n was s t u d i e d q u a n t i t a t i v e l y in i n t a c t ' w i n d o w ' s p r e a d p r e p a r a t i o n s o f mesentery.
Materials and Methods Animals
Male Sprague-Dawley rats (AB Anticimex, Stockholm, Sweden) fed standard pellets for rats and mice (Astra-Ewos AB, S6dert~ilje, Sweden) and water ad libitum were used. The animals were kept in our laboratory for 5 days before experimentation. Five animals per cage were kept in a window-free room with automatic regulation of artificial light (from 06-18 h), temperature (24 _+0.5 ~C) and humidity (60 -+ 2 per cent relative humidity). The animals weighed between 160 and 210 grams at the start of the experiment. As far as possible control animals and treated animals were kept under identical conditions. Care was taken in every experiment to use only animals within a narrow range of weights and to equalise the weights of the animals between the different experimental groups. Microscopic Preparations and Proli[eration Parameters
In the normal rat the mesentery spread preparations are thin and translucent. Both sides of the preparation are covered by a single layer of mesothelial cells enclosing a submesothelial tissue space which contains fibroblastic cells, mast cells, lymphocytes, eosinophilic leucocytes, neutrophilic leucocytes, collagen, elastic fibers and blood vessels. In the present study the pararosaniline-Feulgen reaction for quantitation of DNA, and toluidine blue staining at "pH 4.0 and 0.5" for general cytological morphology and verification of degranulation of mast cells, were employed. The technique for quantitation of cell proliferation parameters has been described in detail elsewhere (Enerb/ick et al. 1976). Estimation of Cell Production. The mitotic index (M.I. = the number of cells in mitosis per 1000 cells) was estimated by counting the number of mitoses among 3,000 or 10,000 randomly chosen mesothelial and fibroblastic cells per animal. DNA Content in IndividualFibroblastic Cells. In one preparation per animal 200 randomly chosen oval fibroblastic nuclei with a smooth nuclear membrane and as a rule, containing one unstained area corresponding to the nucleolus, were measured in Feulgen stained specimens using a 100 x objective. The error of a single observation, e, was less than 3 per cent. Specific DNA Activity in the Mesentery Preparations. 3H-TdR (methyl-3H-thymidine) with a specific activity of 6.7 Ci per mmole (NEN Chemicals GmbH, Dreieichenhain, West Germany) was given as a single intravenous dose of 2 gCi per g body weight (1 mCi/ml) 60 minutes before the animals were killed. The specific DNA activity per specimen (radioactivity expressed as c.p.m, divided by the DNA content of the specimen) was determined by combining Feulgen photometric estimation of the total amount of DNA per specimen and a radiochemical technique using liquid scintillation spectrometry. Two or three mesentery preparations were analysed per animal.
Mast Cell Activation and Tissue Cell Proliferation
291
Drug Used for Mast Cell Degranulation C o m p o u n d 48/80", a condensation product of p-methoxy-N-methyl-phenyl-aethyl-amine with formaldehyde (Paton, 1951) dissolved in saline was injected percutaneously i.p. as a single dose of 1 p.g per g body weight (1 ml solution/100 g) at 9 a.m. The animals were killed 24 and 32h later. Untreated rats killed at 9 a.m. or rats injected with saline (1 ml/100g) at 9 a.m. and killed 24 or 32h later were used as controls. All solutions to be injected were checked for pH 7.2 -+ 0.1, were administered at room temperature and were sterile after Millipore filtration (pore size 0.22 gm).
Statistical Methods The error of a single observation, e, was calculated from paired dependent observations as described by Dahlberg (1948). The significance of the difference between two means was assessed by Student's t-test. The randomization test for two independent samples according to Siegel (1956) was used for comparing the effect of 48/80 versus saline.
Results
Microscopic examination of the mesentery preparations in rats treated with a single intraperitoneal dose of 48/80 showed a mast cell degranulation in toluidine blue preparations, and characteristic tissue changes appeared within 8 to 16 h. Only rarely were degranulated mast cells seen in saline treated animals and hardly ever in untreated controls. The 48/80 treatment was followed by an increased number of fibroblastic cells with strongly basophilic cytoplasm, nuclear polymorphism as in proliferating tissues or cell cultures, a markedly increased number of mitoses (from about 24 h), and an apparent increase in the amount of collagen (Figs. l, 2). Lymphocytes and leucocytes which normally occur patch-wise was only occasionally slightly increased in number. These microscopic alterations persisted throughout the 48 h observation period. The proliferation occurred in fibroblastic as well as in mesothelial cells at apparently the same rate. Quantitative data describing the cell proliferation were obtained 24 and 32 h after a single injection of Compound 48/80. The results of the estimation of specific DNA activity are summarized in Figure 3. At 24 h, the 48/80 treatment caused a statistically significant increase in the specific DNA activity compared to untreated controls (p < 0.003). Compared to saline treated controls this increase was approximately 3 to 4-fold. There was no significant difference between 24 and 32 h after 48/80 treatment. Compared to saline treated animals killed at the same hour of the day the increase was, however, somewhat larger at 32 h after 48/80 treatment than at 24 h (Table 1). By constructing weighted averages and using the randomization test for two independent samples the specific DNA activity was significantly increased by 48/80 compared to saline at 24 and 32 h (p _< 0.01). Another way of estimating DNA synthesis was by determining the DNA content in individual fibroblastic cells. From histograms of the distribution of the DNA content per cell the relative number of cells with the DNA content of the S and G2 phases was established. The results are summarised in Figures 4 and 5 and *
We thank Dr. B. H6gberg, M.D., AB Leo, H~lsingborg, Sweden, for the generous gift of this drug
292
K. Norrby et al.
Fig. 1A and B. Photomicrographs of mesentery spread preparations from untreated rat A and from rat 24 h after a single injection of Compound 48/80 B. Totuidine blue at pH 4. The darkly stained cells in A are mast cells filled with metachromatically stained granules. In B note disappearance of mast cells filled
Mast Cell Activation and Tissue Cell Proliferation
293
with granules and the occurrence of small metachromatic granules distributed between cells and possibly also within the cytoplasm of some fibroblastic and mesothelial cells. Note also cytoplasmic basophilia, nuclear polymorphism and mitosis
294
K. Norrby et al.
Fig. 2A and B. Photomicrographs of Feulgen-stained specimens from mesentery preparations of untreated control rat A and rat 24h after a single injection of 48/80 B. The large and faintly stained
Mast Cell Activation and Tissue Cell Proliferation
295
nuclei belong to mesothelial cells whereas the small and dark oval nuclei belong to fibroblast-like cells. Note polymorphism and mitoses in B
296
K. Norrby et al.
40 m ..-7.
IJJ +l C
m |
30
E
"~
20
E
Z
T
, 0_
m
T__
10
1
o,[ ,,
Fig. 3. The specific DNA activity (mean + S.E.) from 7 untreated controls (C), 6 saline treated controls (S) and 11 animals treated with 48/80. The untreated controls and saline treated and 48/80 treated animals were also killed at 9 a.m. 24 h after treatment. Saline treated and 48/80 treated animals were killed at 5 p.m., 32 h after treatment. Using Student's t-test there was a statistically significant difference between the 48/80 treated animals 24 h after treatment and untreated controls (p < 0.003)~ Using the randomization test for two independent samples and constructing weighted averages of the results from 24 and 32 h it was found that 48/80 caused a statistically significant increase in specific D N A activity compared to saline (p _< 0.01)
co -] c
C
S
4E /80 2 4 hrs
9a.m.
9a.m.
s
48/80 32 hrs 5p.m.
in Tables 1 and 2. The G1 modal distributions were narrow throughout, the S.D. being less than 4 per cent of the mean. Polyploidy except for tetraploid 62 cells -- was not found. As seen from Figures 4 and 5 and Table 2 there was approximately a 5 to 7-fold increase in the number of S and G2 cells in the saline treated controls compared to untreated controls but an additional 3 to 5-fold increase in the 48/80 treated animals. (Proliferation was also induced in mesothelial cells, the relative number of S and G2 cells 32 h after the 48/80 treatment being somewhat less than for the fibroblastic cell population). The relative number of S and G2 fibroblastic cells was higher at 32 h after the 48/80 than at 24 h. Thus the 48/80 treated animals showed approximately a 18 to 26-fold increase in the relative number of S and G2 cells compared to untreated controls. Using the randomization test for two independent samples and constructing weighted averages of the results at 24 and 32 h it was found that 48/80 caused a statistically significant increase (p < 0.01) in the relative number of cells having a DNA-content in the S and G2 regions compared to saline.
Mast Cell Activation and Tissue Cell Proliferation
297
Table 1. Mean percentage ( + S.E.) increase in the three proliferation parameters studied 24 and 32 h after 48/80 treatment with corresponding mean values for saline treated controls set to 100 per cent. (n) = number of animals. By constructing weighted averages of the results (expressed in AU of specific DNA activity and relative number of cells in S and G2 regions) at 24 and 32 h using the randomization test for two independent samples, the increase caused by 48/80 as compared to saline in specific DNA activity as well as in the proportion of S and G2 cells was statistically significant (p _
Cell and Tissue Research ~, by Springer-Verlag 1976
Mast Cell Activation and Tissue Cell Proliferation* Klas Norrby, Lennart EnerNick and Lennart Franz6n** *** Department of Pathology II, University of Link6ping, Sweden
Summary. The effect of mast cell activation and degranulation on the proliferation in the intact mesentery was studied in Sprague-Dawley rats. Mast cell activation was achieved by a single intraperitoneal injection of Compound 48/80. The proliferation was studied using three independent methods for estimation of cell production and D N A synthesis: 1. the mitotic index, 2. the relative number of cells having a D N A content in the S and G2 regions, by Feulgen photometric measurement in individual cells, and 3. the specific D N A activity, employing a method which combines a liquid scintillation technique after an intravenous injection of 3H-thymidine and Feulgen photometric determination of the D N A content per membrane preparation. It was found that the proliferation of the normal mesenchymal cells adjacent to the activated and degranulated mast cells in the mesentery was significantly increased within 24 and 32 h, the maximum increase being more than 20-fold compared to untreated controls. The results suggest that the c o m m o n type of mast cell may have a pathophysiological function related to stimulation of local cell proliferation.
Key words: Mast cell - C o m p o u n d 48/80 - Cell proliferation - Mesentery D N A - Mitosis.
Introduction A hypothetical relationship between mast cells and connective tissue activity has been discussed in the literature for a number of years (Riley, 1959; Selye, 1965; Send offprint requests to: Dr. Klas Norrby, Department of Pathology II, University of Link6ping, S 58185 Link6ping, Sweden
* Supported by grants from the Swedish Medical Research Council (Project 12X-2235) and from the Medical Faculty, Universityof Link6ping ** We thank Brita Saderlund, Margareta Oden6 and Irane Svensson for skilful technical assistance, and Erik Leander, Ph.D., for help with ~tatistical methods *** Part of this work was presented at the 7th Meeting of the European Study Group for Cell Proliferation, 5-9 May 1975, in Amsterdam, The Netherlands
290
K. Norrby et al.
F e r n e x , 1968). It was r e c e n t l y f o u n d in o u r l a b o r a t o r y ( N o r r b y , 1973b) t h a t the m a s t cell c o m p o n e n t s h e p a r i n , h i s t a m i n e a n d s e r o t o n i n at v e r y l o w c o n c e n t r a t i o n s , a l o n e o r m i x e d , release f i b r o b l a s t i c cells f r o m d e n s i t y - d e p e n d e n t i n h i b i t i o n o f cell d i v i s i o n . T h e s e f i n d i n g s in v i t r o s u g g e s t e d t h a t m a s t cell c o m p o n e n t s m i g h t also act as c e l l - d i v i s i o n - s t i m u l a t i n g a g e n t s in v i v o . T h e a i m o f the p r e s e n t w o r k was t o s t u d y the effect o f m a s t cell d e g r a n u l a t i o n o n the p r o l i f e r a t i o n o f a d j a c e n t n o r m a l m e s e n c h y m a l tissue cells. T h e s m a l l - g u t m e s e n t e r y o f t h e rat was c h o s e n as test tissue. M a s t cell d e g r a n u l a t i o n was a c h i e v e d by a single i n j e c t i o n o f C o m p o u n d 48/80. S u c h a t r e a t m e n t r e g u l a r l y i n d u c e d a m a r k e d p r o l i f e r a t i o n o f n e a r - b y f i b r o b l a s t i c a n d m e s o t h e l i a l cells. U s i n g a techn i q u e d e v e l o p e d in this l a b o r a t o r y ( E n e r b / i c k et al., 1976) the i n d u c e d p r o l i f e r a t i v e r e a c t i o n was s t u d i e d q u a n t i t a t i v e l y in i n t a c t ' w i n d o w ' s p r e a d p r e p a r a t i o n s o f mesentery.
Materials and Methods Animals
Male Sprague-Dawley rats (AB Anticimex, Stockholm, Sweden) fed standard pellets for rats and mice (Astra-Ewos AB, S6dert~ilje, Sweden) and water ad libitum were used. The animals were kept in our laboratory for 5 days before experimentation. Five animals per cage were kept in a window-free room with automatic regulation of artificial light (from 06-18 h), temperature (24 _+0.5 ~C) and humidity (60 -+ 2 per cent relative humidity). The animals weighed between 160 and 210 grams at the start of the experiment. As far as possible control animals and treated animals were kept under identical conditions. Care was taken in every experiment to use only animals within a narrow range of weights and to equalise the weights of the animals between the different experimental groups. Microscopic Preparations and Proli[eration Parameters
In the normal rat the mesentery spread preparations are thin and translucent. Both sides of the preparation are covered by a single layer of mesothelial cells enclosing a submesothelial tissue space which contains fibroblastic cells, mast cells, lymphocytes, eosinophilic leucocytes, neutrophilic leucocytes, collagen, elastic fibers and blood vessels. In the present study the pararosaniline-Feulgen reaction for quantitation of DNA, and toluidine blue staining at "pH 4.0 and 0.5" for general cytological morphology and verification of degranulation of mast cells, were employed. The technique for quantitation of cell proliferation parameters has been described in detail elsewhere (Enerb/ick et al. 1976). Estimation of Cell Production. The mitotic index (M.I. = the number of cells in mitosis per 1000 cells) was estimated by counting the number of mitoses among 3,000 or 10,000 randomly chosen mesothelial and fibroblastic cells per animal. DNA Content in IndividualFibroblastic Cells. In one preparation per animal 200 randomly chosen oval fibroblastic nuclei with a smooth nuclear membrane and as a rule, containing one unstained area corresponding to the nucleolus, were measured in Feulgen stained specimens using a 100 x objective. The error of a single observation, e, was less than 3 per cent. Specific DNA Activity in the Mesentery Preparations. 3H-TdR (methyl-3H-thymidine) with a specific activity of 6.7 Ci per mmole (NEN Chemicals GmbH, Dreieichenhain, West Germany) was given as a single intravenous dose of 2 gCi per g body weight (1 mCi/ml) 60 minutes before the animals were killed. The specific DNA activity per specimen (radioactivity expressed as c.p.m, divided by the DNA content of the specimen) was determined by combining Feulgen photometric estimation of the total amount of DNA per specimen and a radiochemical technique using liquid scintillation spectrometry. Two or three mesentery preparations were analysed per animal.
Mast Cell Activation and Tissue Cell Proliferation
291
Drug Used for Mast Cell Degranulation C o m p o u n d 48/80", a condensation product of p-methoxy-N-methyl-phenyl-aethyl-amine with formaldehyde (Paton, 1951) dissolved in saline was injected percutaneously i.p. as a single dose of 1 p.g per g body weight (1 ml solution/100 g) at 9 a.m. The animals were killed 24 and 32h later. Untreated rats killed at 9 a.m. or rats injected with saline (1 ml/100g) at 9 a.m. and killed 24 or 32h later were used as controls. All solutions to be injected were checked for pH 7.2 -+ 0.1, were administered at room temperature and were sterile after Millipore filtration (pore size 0.22 gm).
Statistical Methods The error of a single observation, e, was calculated from paired dependent observations as described by Dahlberg (1948). The significance of the difference between two means was assessed by Student's t-test. The randomization test for two independent samples according to Siegel (1956) was used for comparing the effect of 48/80 versus saline.
Results
Microscopic examination of the mesentery preparations in rats treated with a single intraperitoneal dose of 48/80 showed a mast cell degranulation in toluidine blue preparations, and characteristic tissue changes appeared within 8 to 16 h. Only rarely were degranulated mast cells seen in saline treated animals and hardly ever in untreated controls. The 48/80 treatment was followed by an increased number of fibroblastic cells with strongly basophilic cytoplasm, nuclear polymorphism as in proliferating tissues or cell cultures, a markedly increased number of mitoses (from about 24 h), and an apparent increase in the amount of collagen (Figs. l, 2). Lymphocytes and leucocytes which normally occur patch-wise was only occasionally slightly increased in number. These microscopic alterations persisted throughout the 48 h observation period. The proliferation occurred in fibroblastic as well as in mesothelial cells at apparently the same rate. Quantitative data describing the cell proliferation were obtained 24 and 32 h after a single injection of Compound 48/80. The results of the estimation of specific DNA activity are summarized in Figure 3. At 24 h, the 48/80 treatment caused a statistically significant increase in the specific DNA activity compared to untreated controls (p < 0.003). Compared to saline treated controls this increase was approximately 3 to 4-fold. There was no significant difference between 24 and 32 h after 48/80 treatment. Compared to saline treated animals killed at the same hour of the day the increase was, however, somewhat larger at 32 h after 48/80 treatment than at 24 h (Table 1). By constructing weighted averages and using the randomization test for two independent samples the specific DNA activity was significantly increased by 48/80 compared to saline at 24 and 32 h (p _< 0.01). Another way of estimating DNA synthesis was by determining the DNA content in individual fibroblastic cells. From histograms of the distribution of the DNA content per cell the relative number of cells with the DNA content of the S and G2 phases was established. The results are summarised in Figures 4 and 5 and *
We thank Dr. B. H6gberg, M.D., AB Leo, H~lsingborg, Sweden, for the generous gift of this drug
292
K. Norrby et al.
Fig. 1A and B. Photomicrographs of mesentery spread preparations from untreated rat A and from rat 24 h after a single injection of Compound 48/80 B. Totuidine blue at pH 4. The darkly stained cells in A are mast cells filled with metachromatically stained granules. In B note disappearance of mast cells filled
Mast Cell Activation and Tissue Cell Proliferation
293
with granules and the occurrence of small metachromatic granules distributed between cells and possibly also within the cytoplasm of some fibroblastic and mesothelial cells. Note also cytoplasmic basophilia, nuclear polymorphism and mitosis
294
K. Norrby et al.
Fig. 2A and B. Photomicrographs of Feulgen-stained specimens from mesentery preparations of untreated control rat A and rat 24h after a single injection of 48/80 B. The large and faintly stained
Mast Cell Activation and Tissue Cell Proliferation
295
nuclei belong to mesothelial cells whereas the small and dark oval nuclei belong to fibroblast-like cells. Note polymorphism and mitoses in B
296
K. Norrby et al.
40 m ..-7.
IJJ +l C
m |
30
E
"~
20
E
Z
T
, 0_
m
T__
10
1
o,[ ,,
Fig. 3. The specific DNA activity (mean + S.E.) from 7 untreated controls (C), 6 saline treated controls (S) and 11 animals treated with 48/80. The untreated controls and saline treated and 48/80 treated animals were also killed at 9 a.m. 24 h after treatment. Saline treated and 48/80 treated animals were killed at 5 p.m., 32 h after treatment. Using Student's t-test there was a statistically significant difference between the 48/80 treated animals 24 h after treatment and untreated controls (p < 0.003)~ Using the randomization test for two independent samples and constructing weighted averages of the results from 24 and 32 h it was found that 48/80 caused a statistically significant increase in specific D N A activity compared to saline (p _< 0.01)
co -] c
C
S
4E /80 2 4 hrs
9a.m.
9a.m.
s
48/80 32 hrs 5p.m.
in Tables 1 and 2. The G1 modal distributions were narrow throughout, the S.D. being less than 4 per cent of the mean. Polyploidy except for tetraploid 62 cells -- was not found. As seen from Figures 4 and 5 and Table 2 there was approximately a 5 to 7-fold increase in the number of S and G2 cells in the saline treated controls compared to untreated controls but an additional 3 to 5-fold increase in the 48/80 treated animals. (Proliferation was also induced in mesothelial cells, the relative number of S and G2 cells 32 h after the 48/80 treatment being somewhat less than for the fibroblastic cell population). The relative number of S and G2 fibroblastic cells was higher at 32 h after the 48/80 than at 24 h. Thus the 48/80 treated animals showed approximately a 18 to 26-fold increase in the relative number of S and G2 cells compared to untreated controls. Using the randomization test for two independent samples and constructing weighted averages of the results at 24 and 32 h it was found that 48/80 caused a statistically significant increase (p < 0.01) in the relative number of cells having a DNA-content in the S and G2 regions compared to saline.
Mast Cell Activation and Tissue Cell Proliferation
297
Table 1. Mean percentage ( + S.E.) increase in the three proliferation parameters studied 24 and 32 h after 48/80 treatment with corresponding mean values for saline treated controls set to 100 per cent. (n) = number of animals. By constructing weighted averages of the results (expressed in AU of specific DNA activity and relative number of cells in S and G2 regions) at 24 and 32 h using the randomization test for two independent samples, the increase caused by 48/80 as compared to saline in specific DNA activity as well as in the proportion of S and G2 cells was statistically significant (p _
