Influence of the Beta-Adrenergic Antagonists Propranolol, Practolol and Oxprenolol On the Proliferation of Rat Jejunal Crypt Cells

Abstract. Beta-adrenergic blockade by quite large doses of propranolol, practolol and oxprenolol, once or continuously applied, does not influence jejunal crypt-cell proliferation in the rat. After a single i.p. injection of 20 mg/kg propranolol or practolol and even of 100 mg/kg practolol, the mitotic index, the labelling index and the duration of the S phase do not differ between treated and untreated control animals nor between animals treated with the different drugs. Continuous application of 30 mg/kg/d propranolol, practolol or oxprenolol for 7 or 14 days does not affect the mitotic and labelling indices either, nor does it change the duration of the cycle of the jejunal crypt cells and its phases as determined by the percent labelled mitoses method. These results are in contrast to those reported previously by Tutton & Helme (1974).     

Influence of the beta-adrenergic antagonists propranolol, practolol and oxprenolol on the proliferation of rat jejunal crypt cells

Beta-adrenergic blockade by quite large doses of propranolol, practolol and oxprenolol, once or continuously applied, does not influence jejunal crypt-cell proliferation in the rat. After a single i.p. injection of 20 mg/kg propranolol or practolol and even of 100 mg/kg practolol, the mitotic index, the labelling index and the duration of the S phase do not differ between treated and untreated control animals nor between animals treated with the different drugs. Continuous application of 30 mg/kg/d propranolol, practolol or oxprenolol for 7 or 14 days does not affect the mitotic and labelling indices either, nor does it change the duration of the cycle of the jejunal crypt cells and its phases as determined by the percent labelled mitoses method. These results are in contrast to those reported previously by Tutton & Helme (1974).     

The effect of single and of multiple doses of prednisolone tertiary butyl acetate on cell population kinetics in the small bowel mucosa of the rat.

The effect of single and of multiple doses of prednisolone upon cell population kinetics in the rat jejunal crypt was investigated, using autoradiography and stathmokinetic techniques with vincristine. Single injections of prednisolone (2.5 mg/kg body weight) induced a depression both flash thymidine labelling and mitotic indices; this change was shown to be due to a decreased cell production rate. Recovery of these proliferative indices occurred over seven days after injection; measurement of crypt size parameters showed a transient decrease in crypt population. Multiple daily injections of prednisolone (1 mg/kg body weight) produced a more sustained decrease in labelling and mitotic indices, which lasted as long as injections were continued (7 days); stathmokinetic techniques showed decreases in cell production rates, and the crypt population was also depressed throughout this period. It is concluded that prednisolone depresses cell proliferative rates in rat jejunal mucosa.     

Chronic metoclopramide treatment stimulates T lymphocyte proliferation in the spleen but not in the thymus.

The effect of chronic metoclopramide administration (for 10 days at a daily dose of 5 mg/kg body weight subcutaneously) on cell proliferation in spleen and in thymus was investigated. Cell proliferation was evaluated by the stathmokinetic method with the use of vincristine. It was found that metoclopramide administration results in a statistically significant increase in the value of the mean mitotic activity rate index (MMAR) of splenocytes in the areas around arteries. At the same time no statistically significant changes were demonstrated in the MMAR index values obtained for splenocytes present in the germinal centers of the spleen. No significant changes in the MMAR index could also be found for thymocytes.     

Effects of Aflatoxin B1 Exposure and Sodium Selenite Supplementation on the Histology,Cell Proliferation,and Cell Cycle of Jejunum in Broilers

The aim of this study was to investigate the effects of aflatoxin B₁ (AFB₁) exposure and sodium selenite supplementation on the histology, cell proliferation and cell cycle of jejunum in broilers. A total of 240 1-day-old male AA broilers were divided into four groups of 60 each, fed with basal diet (control group), 0.3 mg/kg AFB₁ (AFB₁ group), 0.4 mg/kg supplement Se (Se group), and 0.3 mg/kg AFB₁?+?0.4 mg/kg supplement Se (AFB₁ + Se group) for 21 days, respectively. Compared with the control group, decreased jejunal villus height, villus height/crypt ratio, and proliferation cell nuclear antigen (PCNA)-positive cells, and G₂/M phase arrest and shedded epithelial cells on the tip of jejunal villus were observed in AFB₁ groups at 7 and 14 days of age. However, the villus/crypt ratio, PCNA-positive cells and cell percentage of G₀/G₁, S, and G₂/M phases had no significant differences between AFB₁ group and control group at 21 days. Simultaneous supplementation with sodium selenite restored these parameters to be close to those in control group. In conclusion, 0.3 mg/kg AFB₁ in the diet inhibits the development of broiler’s jejunum by reducing cellular proliferation and inducing G₂/M arrest during only the first 2 weeks after hatching. Supplementation of dietary sodium selenite at the concentration of 0.4 mg/kg Se had protective action against these toxic effects of AFB₁.     

The decay of autoradiographic grain number over crypt base columnar cells in murine ileum as a measure of their generation time

The decay in the number of grains over [3H]-thymidine labelled crypt base columnar cells (BCC) in autoradiographs of the ileum of BDF1 mice has been studied. The results revealed that using the conventional grain count halving (GCH) method it is possible to obtain an estimation of the generation time (Tc) of the proliferative BCC cells in the Paneth cell zone (PC-zone) of 18.8 +/- 0.74 h. This lies within the range obtained by the percent labelled mitoses (PLM) method, but is shorter than most values obtained by stathmokinetic methods. The present data show no evidence for a shortening of the cell cycle 3 days after irradiation (8 Gy) which is contrary to some earlier observations. Some reasons for this discrepancy are discussed. The comparatively high labelling index of the BCC allows a larger amount of data to be easily collected, compared with the PLM technique, and correction factors which take into account the complicated shape of the bottom of the crypt are not required.     

Cell population kinetics in the rat jejunal crypt.

Cell kinetics in the jejunal crypt of the male Wistar rat were studied using autoradiographic techniques with tritiated thymidine and a stathmokinetic technique with vincristine. The migration rate measured by following the movement of the 50% peak on the labelling index distribution curve with time after injection of tritiated thymidine gave a value of 1-43 +/- 0-14 (SE) cell positions per hour, compared with a value from a cumulative birth rate of 1-78 cell positions per hour. Tht crypt column length was 32-9 +/- 0-2 cells and the column count was 22-3 +/- 0-2. This measurement gave a total crypt population of 734 cells, compared with an estimate of 650 +/- l from direct observation of squashed, microdissected crypts. In each crypt 22-5 +/- 0-5 mitoses were present, and the crypt cell production rate was 32 cells per crypt per hour; this latter value was confirmed using two independent techniques. The crypt growth fraction calculated from the durations of phases of the cell cycle and the labelling index was 0-62. A value of 0-61 was found from the labelling index distribution curve. As assessed from crypt squashes, there were 403 proliferating cells per crypt.     

CELL POPULATION KINETICS IN THE RAT JEJUNAL CRYPT

Cell kinetics in the jejunal crypt of the male Wistar rat were studied using autoradiographic techniques with tritiated thymidine and a stathmokinetic technique with vincristine. The migration rate measured by following the movement of the 50 % peak on the labelling index distribution curve with time after injection of tritiated thymidine gave a value of 1.43 ± 0.14 (SE) cell positions per hour, compared with a value from a cumulative birth rate of 1.78 cell positions per hour. The crypt column length was 32.9 ± 0.2 cells and the column count was 22.3 ± 0.2. This measurement gave a total crypt population of 734 cells, compared with an estimate of 650 ± 6 from direct observation of squashed, microdissected crypts. In each crypt 22.5 ± 0.5 mitoses were present, and the crypt cell production rate was 32 cells per crypt per hour; this latter value was confirmed using two independent techniques. The crypt growth fraction calculated from the durations of phases of the cell cycle and the labelling index was 0.62. A value of 0.61 was found from the labelling index distribution curve. As assessed from crypt squashes, there were 403 proliferating cells per crypt.     

Effect of cyclophosphamide on the proliferation of L 1210 ascites tumour cells and of jejunal crypt cells in the mouse

Cyclophosphamide (CY) does not act in a cell-cycle specific manner, i.e. exclusively on proliferating cells. It also kills non-proliferating cells, as shown by application of CY to L 1210 ascites tumour-bearing mice during plateau phase growth of the tumour. Moreover, treatment with CY of L 1210 ascites tumour cells, double-labelled with [3H] and [14C]-thymidine, suggests that CY is not cell cycle phase dependent, but kills cells out of all cycle phases. There is also an extensive cytocidal effect of CY (300 mg/kg) on the jejunal crypt cells of the mouse, which is even more pronounced than that of cisplatinum (DDP, 13 mg/kg). However, rapid regeneration of crypt cells occurs after treatment with the drugs.     

Protection by WR-3689 against gamma-ray-induced intestinal damage: comparative effect on clonogenic cell survival, mouse survival, and DNA damage

The aminophosphorothioate WR-3689 was characterized for its ability to protect mouse jejunal cells in vivo from single doses of X or gamma radiation. First, the effect of the drug on the survival of jejunal stem cells was examined using a clonogenic end point, the crypt microcolony assay. When WR-3689 was administered 30 min prior to whole-body irradiation, the number of surviving crypt cells was markedly increased at all doses of the drug, although protection began to level out at doses larger than 600 mg/kg. Protection was maximal when the drug was given 30 min before whole-body irradiation and declined rapidly with both shorter and longer intervals. Protection factors (PFs) were obtained by measuring survival curves for clonogenic crypt cells as a function of radiation dose; WR-3689 given 30 min before whole-body irradiation protected jejunum in the microcolony assay with a PF of 1.26 +/- 0.02, 1.50 +/- 0.10, and 1.65 +/- 0.10 at doses of 200, 400, and 800 mg/kg, respectively. Next, the effect of WR-3689 on the survival of jejunal stem cells was determined by assaying the survival of mice given X-ray doses to the whole abdomen in the range leading to death from the gastrointestinal syndrome. The PFs based on the LD50 values for 11-day survival were 1.31 +/- 0.05 (200 mg/kg) and 1.48 +/- 0.05 (400 mg/kg). Crypt-cell survival and animal survival were thus modified to a similar extent by this agent. Finally, the effect of WR-3689 on the induction of DNA single-strand breaks (SSBs) in jejunal cells was measured using an adaptation of the alkaline elution methodology. In mice treated with WR-3689 (400 or 800 mg/kg) 30 min prior to whole-body irradiation with 10 Gy there was no significant reduction in the number of DNA SSBs induced either in samples of the jejunum or in the cycling crypt cells, providing further evidence that there is no simple relationship between the modification of DNA SSBs and the survival of jejunal stem cells.     

Acute and subacute effects of thiamine deficiency on the morphometry and cell kinetics of jejunal and ileal epithelial cells

The effects of acute and subacute thiamine deficiency on jejunal and ileal epithelial cells were studied in rats, using crypt and villus cell population, crypt cell production per crypt (CCPC), crypt growth fraction (Ip) and crypt cell cycle time (Tc) as parameters. In acute thiamine deficiency there was marked jejunal hypoplasia of the crypt and villus, but in the ileum there was hypoplasia only of the crypt. The jejunal epithelium of the subacute thiamine deficiency (STD) group showed no morphometric changes. In contrast, in the ileal epithelium of STD rats there was decreased crypt depth and villus cell population. Thiamine deficiency had no significant effect on CCPC, Ip and Tc.     

Effect of Cyclophosphamide On the Proliferation of L 1210 Ascites Tumour Cells and of Jejunal Crypt Cells In the Mouse

Abstract. Cyclophosphamide (CY) does not act in a cell-cycle specific manner. i.e. exclusively on proliferating cells. It also kills non-proliferating cells, as shown by application of CY to L 1210 ascites tumour-bearing mice during plateau phase growth of the tumour. Moreover, treatment with CY of L 1210 ascites tumour cells, double-labelled with [³Hl and [¹⁴C]-thymidine, suggests that CY is not cell cycle phase dependent, but kills cells out of all cycle phases.
There is also an extensive cytocidal effect of CY (300 mg/kg) on the jejunal crypt cells of the mouse, which is even more pronounced than that of cisplatinum (DDP, 13 mg/kg). However, rapid regeneration of crypt cells occurs after treatment with the drugs.     

Effects of spaceflight on the proliferation of jejunal mucosal cells

The mitotic indices, villus heights, and crypt depths were determined in each of three jejunal regions (proximal, middle, and distal) for five animals each in the flight, vivarium, and synchronous groups. Because of the rapid turnover of intestinal mucosal cells and the delay in recovering the flight animals, it is not known whether the proliferation of jejunal mucosal cells is affected by microgravity conditions associated with spaceflight. However, since there were no consistent differences between animals in the flight group and those in the synchronous and vivarium control groups, it appears that any effects of microgravity on the turnover of jejunal mucosal cells are short-lived. Thus, this study represents an initial step in determining the effects of microgravity on the proliferation and turnover of intestinal mucosal cells.     

Analysis of intestinal cell proliferation after guanethidine-induced sympathectomy. II. Percentage labelled mitoses studies

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (Tc) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G1, S and G2 phases of the crypt cell cycle are altered by sympathectomy, G1 accounting for most of the increase in Tc. In addition, the percentage of [3H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.     

Acute and Subacute Effects of Thiamine Deficiency On the Morphometry and Cell Kinetics of Jejunal and Ileal Epithelial Cells

Abstract. The effects of acute and subacute thiamine deficiency on jejunal and ileal epithelial cells were studied in rats, using crypt and villus cell population, crypt cell production per crypt (CCPC), crypt growth fraction (I_p) and crypt cell cycle time (T_c) as parameters. In acute thiamine deficiency there was marked jejunal hypoplasia of the crypt and villus, but in the ileum there was hypoplasia only of the crypt. the jejunal epithelium of the subacute thiamine deficiency (STD) group showed no morphometric changes. In contrast, in the ileal epithelium of STD rats there was decreased crypt depth and villus cell population. Thiamine deficiency had no significant effect on CCPC, I_p and T_c.     

Influence of beta-adrenergic antagonists on cell proliferation rates in the kidney of untreated and diethylnitrosamine-treated male F344 rats.

Some nongenotoxic chemicals which cause kidney tumors have been shown to stimulate tubular cell proliferation. The aim of this study was to evaluate the effects of two beta-adrenoreceptor blocking agents, propranolol and atenolol, on cell proliferation rates in the kidneys of male F344 rats. Immunohistochemical expression of proliferating cell nuclear antigen (PCNA) and mitotic index have been examined in formalin-stored kidneys from F344 rats used in an initiation-promotion study of carcinogenesis. Cell proliferation rate was quantified in the proximal tubule epithelium. Non-initiated rats and rats initiated with a single dose of diethylnitrosamine (DEN, 200 mg/kg, i.p.) were continuously treated with propranolol (75-100 mg/kg) or atenolol (300 mg/kg) by gavage and were sacrificed after 2, 4, 8 or 21 months of experimentation. There were two control groups, one untreated (D1) and one given distilled water by gavage (D1). Control group D1 showed significantly lower cell proliferation rates than the D0 group. In non-initiated rats, propranolol had a weak enhancing effect on cell proliferation, most evident after 4 months, while atenolol had a clear enhancing effect most evident after 8 months of promoting regimen. Treatment with DENalone resulted in a significant increase in cell proliferation rate as compared to group D1. In DEN-initiated rats given propranolol, there was a borderline significant increase in cell proliferation rates, compared to rats given DEN alone, after 8 months of promoting regimen. Atenolol had no effect. Because of the differences in body weight gain and food consumption observed among the various groups, it is suggested that the state of nutrition may have obscured the effects of beta-blockers on cell proliferation rates.     

Neural stimulation of mitotic activity in the crypts of lieberkühn in rat jejunum.

The influence of nerve stimulation and sham-stimulation on the mitotic rate in epithelial cells lining the crypts of Lieberkühn in the jejunum of anaesthetized rats was studied. Administration of the anaesthetic and opening the abdominal cavity was without significant effect on the crypt cell mitotic rate. However, externalizing a loop of jejunum and applying sham-stimuli to its mesenteric nerves resulted in a significant decrease in the crypt cell mitotic rate in that loop. Application of electrical stimuli to the mesenteric nerves of another externalized jejunal loop resulted in a significant increase in the mitotic rate in the crypt cells of that segment. Similar acceleration of crypt cell proliferation by electrical stimuli applied to mesenteric nerves was also seen in chemically sympathectomized rats.     

NEURAL STIMULATION OF MITOTIC ACTIVITY IN THE CRYPTS OF LIEBERKÜHN IN RAT JEJUNUM

The influence of nerve stimulation and sham-stimulation on the mitotic rate in epithelial cells lining the crypts of Lieberkühn in the jejunum of anaesthetized rats was studied. Administration of the anaesthetic and opening the abdominal cavity was without significant effect on the crypt cell mitotic rate. However, externalizing a loop of jejunum and applying sham-stimuli to its mesenteric nerves resulted in a significant decrease in the crypt cell mitotic rate in that loop. Application of electrical stimuli to the mesenteric nerves of another externalized jejunal loop resulted in a significant increase in the mitotic rate in the crypt cells of that segment. Similar acceleration of crypt cell proliferation by electrical stimuli applied to mesenteric nerves was also seen in chemically sympathectomized rats.     

Cell kinetic analysis of a murine macrophage cell line

For the present study, which was performed to find a reliable method suitable for determination of the cell kinetic parameters of a continuous cell line, use was made of the macrophage cell line J774.1. The doubling time of the cell population was approximately 27 h. The continuous labeling curve showed that all the cells divide and almost no quiescent cells occur. The cell-cycle time as determined from the curve of the labeled cells in mitosis, the course of the stathmokinetic index, and time-lapse videorecordings, was about 19 h. The discrepancy between the population doubling time and the cell-cycle time must be due to death and disintegration of cells during culture in vitro. The results indicate that the doubling time of a cell population is not a reliable parameter to determine the kinetics of a population of continuously proliferating cells and that determination of the course of the stathmokinetic index offers a rapid and simple method to establish the cell-cycle time reliably.     

Radioprotection of mouse jejunum by WR-2721 and WR-1065: effects on DNA strand-break induction and rejoining

WR-2721 and its free-thiol metabolite WR-1065 have been characterized for their ability to protect mouse jejunal cells in vivo from the damaging effects of gamma rays with respect to both cytotoxicity and DNA single-strand break (SSB) induction. SSBs were measured both in the whole jejunal epithelium and in the proliferating crypt cells using an adaptation of the alkaline elution methodology. Protection factors (PFs) were also obtained using the microcolony assay for jejunal crypts. In mice treated with WR-1065 (400 mg/kg) 15 or 30 min prior to irradiation, there was a slight but significant reduction in the initial number of SSBs both in the whole jejunum (PF of between 1.17 and 1.22) and in the proliferating crypt cells (PF of between 1.13 and 1.28). At a dose of 200 mg/kg, the PF for SSBs in the proliferating crypt cells was 1.12 +/- 0.07 while that for crypt-cell survival was approximately 2.0. In mice treated with WR-2721 (400 mg/kg) 15 min prior to irradiation, there was little effect on the initial number of SSBs induced both in the whole jejunum (PF of 1.07 +/- 0.11) and in the proliferating crypt cells (PF of 1.04 +/- 0.07). WR-2721 protected jejunum in the microcolony assay with a much greater PF of 1.8. For each drug the PF for SSBs was therefore always much lower than that indicated by the biological end point under identical conditions. Both drugs also retarded the rate of SSB rejoining in each population of cells. These data suggest that mechanisms such as free-radical scavenging by these drugs may contribute to but not completely explain their protective action. Comparison with data obtained previously with cultured CHO cells supports the idea that the action of these drugs at the DNA lesion level may not be dose-modifying, but may also result in a shift in the spectrum of lesions induced by the radiation.