Path. Res. Pract. 188,663-667 (1992)
In Situ Analysis of Adenohypophysis Proliferative Activity in Beagle Dogs Preliminary Results M. Kolopp, G. Poirel, P. Vit and E. Perentes Sandoz Pharma Ltd, Drug Safety Assessment, Department of Toxicology, Basle, Switzerland
SUMMARY In order to establish control data on adenohypophysis cell cycle in beagle dogs, two different approaches were used. In the first approach, the adenohypophysis mitotic index (percentage of cells in mitosis: MI) was determined using pituitary glands from 50 control beagle dogs, 10 to 25 months old, from retrospective regulatory safety assessment studies. We founil that the MI in males was 0.024 ± 0.007 % and in females 0.068 ± 0.019 %. The second approach involved the determination of the adenohypophysis cell fraction in S phase by means of bromodeoxyuridine (BrdU) anti-BrdU method in 16 control beagle dogs, 8 to 20 months old, from ongoing safety assessment studies. We found that the BrdU labeling index (percentage of BrdU-positive cells per total number of cells: BrdU LJ) was 0.053 ± 0.009 % in males and 0.059 ± 0.015 % in females. Analysis of our data indicated that the results obtained from the BrdU LI were more reliable (coefficient of variation rCV) 1.4). No clear difference was found between male and female beagle dogs, regarding adenohypophysis cell proliferation. In both studies, adenohypophysis cell proliferation was found to be age-dependent, with the highest value in immature dogs. No correlation between estrus cycle in females and adenohypophysis cell proliferation was noted.
Introduction
Although the mechanism by which cellular proliferation becomes involved in carcinogenesis is not known, its general importance is recognized. Excessive cellular proliferation appears to be a common feature in tumor induction by non-genotoxic carcinogens which act by cytotoxicity followed by regeneration, or by hormone mimetic action 1. Therefore, it is obvious that early identification of cell cycle modifications in laboratory animals treated with test substances is of major importance in safety assessment of drugs and chemicals. In routine histopathology, it is difficult to estimate the cellular turnover based on morphological criteria only, © 1992 by Gustav Fischer Verlag, Stuttgart
and therefore a clear distinction between normal, hyperplastic and neoplastic tissues sometimes becomes arbitrary. Cell cycle modifications of adenohypophysis are frequent in rodents in long-term safety studies 2 , yet only preliminary control data on adenohypophysis cell cycle have been reported in rats and mice, whereas no data are available for dogs. In male rats, the MI of the adenohypophysis (0.07 % or 6.6 mitoses per mm2 ) is lower than in other organs S, and seems to be related to a rhythmic hypothalamic activity. The MI of pituitary cells has been reported to be age-dependentS (maximum in young rats), sex-dependentS, 9,19 (maximum in females) and, in females, estrus cycle-dependentS (maximum during estrus). The cell types in mitosis have been determined in rats of different 0344-0338/92/0188-0663$3.5 0/0
664 . M. Kolopp et al.
ages and physiological statusJO,12, in rats treated with various compounds 6, 13 and under a number of experimental conditions 11, 16. In this paper we present and discuss our results on adenohypophysis cell proliferation in control beagle dogs, tak!n~. into account both individual and physiological v~n~tIons ..The MI and the BrdU LI were calculated using pItUItary tIssue from 50 and 16 control beagle dogs respectively.
Material and Methods
Mitotic Index Pituitary glands from 25 male and 25 female control beagle dogs, 10 to 25 months old (see Table 1), from eight regulatory safety studies, were fixed in 10 % neutral buffered formalin and embedded in paraffin wax. Five-micron-thick sections of the pituitary glands were cut in ribbons: 15 histological slides were prepared, each with five successive sections. Three slides per pituitary gland were stained with periodic-acid-Schiff (PAS), three according to Nissl, and one slide was stained with hematoxylin and eosin (H&E). Sections were examined by light microscopy (final magnification x 630). Cells in mitosis were counted in six non-successive PAS stained sections (16 optical fields per section). Two to three thousand cells were counted from each adenohypophysis. Histologic sections from ovaries, uteri and mammary glands were also examined in order to evaluate the estrus cycle stage 111 female dogs. Statistical evaluation of the results and comparisons between male and female groups were performed using the unpaired Student's t-test. The results were expressed as mean ± SEM (standard error of the mean). The standard deviation (SD) and coefficient of variation (CV: ratio between SD and mean of group) were calculated for each group.
PAP-complex (Dako Co, lot 098) (dilution 1: 200, 20 min at room temperature). The reaction was developed in freshly prepared 3,3' -diaminobenzidine tetrahydrochloride (Amersham Laboratories, Buckinghamshire, England, lot 22), 5.0 mg in 6.5 ml of 0.05 M Tris-buffered saline (pH. 7.6) containing 0.015 % of hydrogen peroxide. Prior to the treatment with the primary antibody, the sections were saturated with 10 % normal swine serum (Dako Co, lot 010). All reagents were dissolved in 0.05 M Tris-buffered saline (pH 7.6) containing normal swine serum. Negative controls were obtained either by incubation with normal mouse serum, or by omirting the primary antibody. Sections from intestinal mucosa, present on each slide, served as positive controls. All sections were lightly counterstained with hematoxylin. Three slides (nine sections) from each adenohypophysis were prepared for the evaluation of the BrdU LI. The number of immunolabeled cells was counted on a maximum of six nonsuccessive sections using an eyepiece with a test grid (final magnification x 400). The total number of cells counted for each adenohypophysis varied from 7,000 to 13,000. The BrdU LI was calculated for each adenohypophysis as the percentage of BrdUlabeled nuclei among the total number of nuclei scored, excluding those of the vascular components and hematogenous cells. Statistical comparisons between male and female groups were performed as described previously.
Table 1. Adenohypophysis mitotic index (%) of control beagle dogs Age (months)
Male
Female
10-11
0.039 0.044
0.049 0.130
13-17
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.032 0.040 0.041 0.048 0.068 0.108 0.130
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.036 0.043 0.075 0.078 0.080 0.085 0.104 0.152 0.196 0.442
24-25
0.000 0.000 0.000 0.000 0.041
0.000 0.000 0.043 0.087 0.099
0.024 ±0.007
0.068 ±0.019
SD
0.035
0.095
CV
1.46
1.40
Bromodeoxyuridine Labeling Index This evaluation was performed in eight male and eight female control beagle dogs. Their age at necrospy varied from 8 to 20 months (Table 2). Ten milligrams per kilogram of 5-bromo-2' -deoxyuridine (Sigma, St Louis, MO, USA, lot 69F-0449), in 0.9 % NaCI, was administered intravenously thirty minutes before sacrifice. Tissue specimens from the pituitary gland and the small intestine were fixed in 70 % chilled ethanol for 24 h at 4°C embedded in paraffin wax, and 4-5 ~tm serial sections were c~t. Five ribbons with three successive sections each were mounted on glass slides for immunohistochemistry. Three slides from each pituitary gland (three serial sections each) were immunostained using an antiBrdU monoclonal antibody. Sections were first deparaffinized in xylene, rehydrated through graded ethanol to water and the endogenous peroxidase activity was blocked with hydrogen peroxide, 0.5 % in methanol for 30 min. After incubation for 30 min in 2N HCI (Merck, Darmstadt, FRG), the sections were neutralized with 0.1 M Na2B407 (Borax, Merck) for 3 min. The peroxidase-antiperoxidase (PAP) method!7 used sequentially: mouse anti-BrdU monoclonal antibody (Beckton Dickinson, Mountain View, Calif., USA, lot MO 150) (dilution 1: 300, 18 h at 4°C); rabbit anti-mouse immunoglobulins (Dako Co, Santa Barbara, Calif., USA, lot 089) (dilution 1: 50, 15 min at room temperature); swine anti-rabbit immunoglobulins (Dako Co, lot 037) (dilution 1: 50, 15 min at room temperature) and rabbit
Mean ± SEM
Adenoh ypophysis Proliferation . 665
Results
Mitotic Index EV111uation (Table 1) The MI of control beagle dogs was 0.024 ± 0.007 % in males and 0.068 ± 0.019 % in females . This difference was statistically significant (p
In Situ Analysis of Adenohypophysis Proliferative Activity in Beagle Dogs Preliminary Results M. Kolopp, G. Poirel, P. Vit and E. Perentes Sandoz Pharma Ltd, Drug Safety Assessment, Department of Toxicology, Basle, Switzerland
SUMMARY In order to establish control data on adenohypophysis cell cycle in beagle dogs, two different approaches were used. In the first approach, the adenohypophysis mitotic index (percentage of cells in mitosis: MI) was determined using pituitary glands from 50 control beagle dogs, 10 to 25 months old, from retrospective regulatory safety assessment studies. We founil that the MI in males was 0.024 ± 0.007 % and in females 0.068 ± 0.019 %. The second approach involved the determination of the adenohypophysis cell fraction in S phase by means of bromodeoxyuridine (BrdU) anti-BrdU method in 16 control beagle dogs, 8 to 20 months old, from ongoing safety assessment studies. We found that the BrdU labeling index (percentage of BrdU-positive cells per total number of cells: BrdU LJ) was 0.053 ± 0.009 % in males and 0.059 ± 0.015 % in females. Analysis of our data indicated that the results obtained from the BrdU LI were more reliable (coefficient of variation rCV) 1.4). No clear difference was found between male and female beagle dogs, regarding adenohypophysis cell proliferation. In both studies, adenohypophysis cell proliferation was found to be age-dependent, with the highest value in immature dogs. No correlation between estrus cycle in females and adenohypophysis cell proliferation was noted.
Introduction
Although the mechanism by which cellular proliferation becomes involved in carcinogenesis is not known, its general importance is recognized. Excessive cellular proliferation appears to be a common feature in tumor induction by non-genotoxic carcinogens which act by cytotoxicity followed by regeneration, or by hormone mimetic action 1. Therefore, it is obvious that early identification of cell cycle modifications in laboratory animals treated with test substances is of major importance in safety assessment of drugs and chemicals. In routine histopathology, it is difficult to estimate the cellular turnover based on morphological criteria only, © 1992 by Gustav Fischer Verlag, Stuttgart
and therefore a clear distinction between normal, hyperplastic and neoplastic tissues sometimes becomes arbitrary. Cell cycle modifications of adenohypophysis are frequent in rodents in long-term safety studies 2 , yet only preliminary control data on adenohypophysis cell cycle have been reported in rats and mice, whereas no data are available for dogs. In male rats, the MI of the adenohypophysis (0.07 % or 6.6 mitoses per mm2 ) is lower than in other organs S, and seems to be related to a rhythmic hypothalamic activity. The MI of pituitary cells has been reported to be age-dependentS (maximum in young rats), sex-dependentS, 9,19 (maximum in females) and, in females, estrus cycle-dependentS (maximum during estrus). The cell types in mitosis have been determined in rats of different 0344-0338/92/0188-0663$3.5 0/0
664 . M. Kolopp et al.
ages and physiological statusJO,12, in rats treated with various compounds 6, 13 and under a number of experimental conditions 11, 16. In this paper we present and discuss our results on adenohypophysis cell proliferation in control beagle dogs, tak!n~. into account both individual and physiological v~n~tIons ..The MI and the BrdU LI were calculated using pItUItary tIssue from 50 and 16 control beagle dogs respectively.
Material and Methods
Mitotic Index Pituitary glands from 25 male and 25 female control beagle dogs, 10 to 25 months old (see Table 1), from eight regulatory safety studies, were fixed in 10 % neutral buffered formalin and embedded in paraffin wax. Five-micron-thick sections of the pituitary glands were cut in ribbons: 15 histological slides were prepared, each with five successive sections. Three slides per pituitary gland were stained with periodic-acid-Schiff (PAS), three according to Nissl, and one slide was stained with hematoxylin and eosin (H&E). Sections were examined by light microscopy (final magnification x 630). Cells in mitosis were counted in six non-successive PAS stained sections (16 optical fields per section). Two to three thousand cells were counted from each adenohypophysis. Histologic sections from ovaries, uteri and mammary glands were also examined in order to evaluate the estrus cycle stage 111 female dogs. Statistical evaluation of the results and comparisons between male and female groups were performed using the unpaired Student's t-test. The results were expressed as mean ± SEM (standard error of the mean). The standard deviation (SD) and coefficient of variation (CV: ratio between SD and mean of group) were calculated for each group.
PAP-complex (Dako Co, lot 098) (dilution 1: 200, 20 min at room temperature). The reaction was developed in freshly prepared 3,3' -diaminobenzidine tetrahydrochloride (Amersham Laboratories, Buckinghamshire, England, lot 22), 5.0 mg in 6.5 ml of 0.05 M Tris-buffered saline (pH. 7.6) containing 0.015 % of hydrogen peroxide. Prior to the treatment with the primary antibody, the sections were saturated with 10 % normal swine serum (Dako Co, lot 010). All reagents were dissolved in 0.05 M Tris-buffered saline (pH 7.6) containing normal swine serum. Negative controls were obtained either by incubation with normal mouse serum, or by omirting the primary antibody. Sections from intestinal mucosa, present on each slide, served as positive controls. All sections were lightly counterstained with hematoxylin. Three slides (nine sections) from each adenohypophysis were prepared for the evaluation of the BrdU LI. The number of immunolabeled cells was counted on a maximum of six nonsuccessive sections using an eyepiece with a test grid (final magnification x 400). The total number of cells counted for each adenohypophysis varied from 7,000 to 13,000. The BrdU LI was calculated for each adenohypophysis as the percentage of BrdUlabeled nuclei among the total number of nuclei scored, excluding those of the vascular components and hematogenous cells. Statistical comparisons between male and female groups were performed as described previously.
Table 1. Adenohypophysis mitotic index (%) of control beagle dogs Age (months)
Male
Female
10-11
0.039 0.044
0.049 0.130
13-17
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.032 0.040 0.041 0.048 0.068 0.108 0.130
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.036 0.043 0.075 0.078 0.080 0.085 0.104 0.152 0.196 0.442
24-25
0.000 0.000 0.000 0.000 0.041
0.000 0.000 0.043 0.087 0.099
0.024 ±0.007
0.068 ±0.019
SD
0.035
0.095
CV
1.46
1.40
Bromodeoxyuridine Labeling Index This evaluation was performed in eight male and eight female control beagle dogs. Their age at necrospy varied from 8 to 20 months (Table 2). Ten milligrams per kilogram of 5-bromo-2' -deoxyuridine (Sigma, St Louis, MO, USA, lot 69F-0449), in 0.9 % NaCI, was administered intravenously thirty minutes before sacrifice. Tissue specimens from the pituitary gland and the small intestine were fixed in 70 % chilled ethanol for 24 h at 4°C embedded in paraffin wax, and 4-5 ~tm serial sections were c~t. Five ribbons with three successive sections each were mounted on glass slides for immunohistochemistry. Three slides from each pituitary gland (three serial sections each) were immunostained using an antiBrdU monoclonal antibody. Sections were first deparaffinized in xylene, rehydrated through graded ethanol to water and the endogenous peroxidase activity was blocked with hydrogen peroxide, 0.5 % in methanol for 30 min. After incubation for 30 min in 2N HCI (Merck, Darmstadt, FRG), the sections were neutralized with 0.1 M Na2B407 (Borax, Merck) for 3 min. The peroxidase-antiperoxidase (PAP) method!7 used sequentially: mouse anti-BrdU monoclonal antibody (Beckton Dickinson, Mountain View, Calif., USA, lot MO 150) (dilution 1: 300, 18 h at 4°C); rabbit anti-mouse immunoglobulins (Dako Co, Santa Barbara, Calif., USA, lot 089) (dilution 1: 50, 15 min at room temperature); swine anti-rabbit immunoglobulins (Dako Co, lot 037) (dilution 1: 50, 15 min at room temperature) and rabbit
Mean ± SEM
Adenoh ypophysis Proliferation . 665
Results
Mitotic Index EV111uation (Table 1) The MI of control beagle dogs was 0.024 ± 0.007 % in males and 0.068 ± 0.019 % in females . This difference was statistically significant (p
