Focal Suppression and Induction 01 Hyperplasia by the Bladder Carcinogens Butyl(4hydroxybutyl)nitrosamine and Butyl(3-carboxypropyl)nitrosamine in Organ-Cultured Rat Bladder Epithelium 1 David H. Reese, 2 Rosalind D. Friedman, 2 William Gaffield, 3 and Larry K. Keefer 4 , 5 , 6
ABSTRACT- The effects of the bladder carcinogens butyl(4-hydroxybutyl)nitrosamine (BBN) and butyl(3-carboxypropyl)-nitrosamine (BCPN) on proliferating transitional rat epithelium in organ culture were studied. At low to intermediate concentration ranges (0.5-2.9 mM), both compounds appeared to stimulate hyperplasia in some regions of epithelia. The major effect of both carcinogens, however, was to suppress hyperplasia in other regions of epithelia and, at higher concentrations (5-6 mM), to cause necrosis. For comparableconcentrations, BBN was more effective in suppressing proliferation and causing necrosis than was BCPN. - J Natl Cancer Inst 60: 219-223, 1978.
When administered orally in the drinking water, BBN induces bladder cancer in rats (1) and mice (2). BBN is not found in the urine (3), presumably because it is metabolized in the liver. Its principal urinary metabolite has been identified as BCPN (3), which, when administered orally, has also been shown to be selectively carcinogenic to the bladder epithelium (4). Both BBN and BCPN are carcinogenic when administered by intravesicular instillation (5). Presumably the transitional epithelial cells have the ability to metabolize BBN to BCPN and, subsequently, BCPN to the ultimate carcinogen. Because little is known about the early effects and mode of action of BBN and BCPN and in view of the apparent ability of both carcinogens to act on the bladder epithelium without prior activation in other organs, studies were initiated to determine what effects BBN and BCPN might have on the bladder epithelium during early periods of exposure to these carcinogens. Since we were primarily interested in the early effects of the carcinogens on the histologic organization of the epithelium, the analysis was done in organ culture because it allows experimentation under carefully controlled and defined conditions on epithelia that are structurally intact and thus more characteristic of normal bladder epithelium than are cell cultures. By characterizing the effect of BBN and BCPN in culture, we hope to make some tentative conclusions concerning the initial effects that these carcinogens have on the epithelium in vivo. This study describes the effects of BBN and BCPN on epithelia cultured under conditions that stimulate proliferation. The effects of these carcinogens on nonproliferating transitional epithelium will be considered later.
MATERIALS AND METHODS Male 6- to 7-week-old Fischer rats were obtained VOL. 60, NO. I, JANUARY 1978
219
from the Charles River Breeding Laboratories , North Wilmington, Massachusetts. The organ-culture procedure was described in (6). In the present experiments, Waymouth's MB 752/1 medium (7) was used and cultures were maintained at 35.5° C. As in previous studies (6, 8), serum or other protein supplements were not used. Before BBN was added to the culture medium, it was dissolved in absolute ethanol. Ethanol was added to BCPN-containing medium and control medium so that all media contained the same ethanol concentrations (0.5%). All preparations of culture media were sterilized by being passed through 0.45- JL pore-size membrane filters (Millipore Corp., Bedford, Mass.). Tissues were exposed to carcinogen for the entire 7-day culture period and medium was changed at 2-day intervals. We assessed the effect of the two carcinogens on the bladder epithelium by determining the extent to which they affected the number of cells in the epithelium. We accomplished this by scoring the number of nuclei per unit length (150 JL) of epithelium from 3 or 4 organ cultures for each experimental group. This procedure was detailed in (6). BCPN (melting point, 39-40° C) was synthesized as described in (9). BBN [boiling point, 125-127° C (0.2 mm Hg)] was synthesized by Dr. EImer Reist, Stanford Research Institute, Palo Alto, California, using the method of Schmähl and Krüger (10) and was obtained from the National Cancer Institute Chemical Repository (Dr. David Longfellow, Project Officer). To demonstrate the stability of BBN and BCPN in culture, we did stability studies on both compounds, in the absence of bladder tissues, under exaggerated conABBREVIATIONS USED: BBN = butyl(4-hydroxybutyl)nitrosamine; BCPN = butyl(3-carboxypropyl)nitrosamine; m]e = mass divided by elementary charge; H & E = hematoxylin and eosin.
Received May 17, 1977; accepted August 18, 1977. Lung Cancer Branch, National Cancer Institute (NCI), National Institutes of Health, Public Health Service, V.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 Western Regional Research Center, Agricultural Research Service, V.S. Department of Agriculture, Berkeley, Calif. 94710. 4 Analytical Chemistry Section, Carcinogen Metabolism and Toxicology Branch, NCI. 5 Address reprint requests to Dr. Keefer. 6 We thank Dr. John Driscoll, Division of Cancer Treatment, NCI, for help in predicting partition coefficients, Mr. J. Richard Miller, Carcinogen Metabolism and Toxicology Branch, NCI, for the mass spectrometry measurements, and Ms. Margaret O'Boyle, Lung Cancer Branch, NCI, for typing the manuscript. 1
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ditions of time and temperature. We determined the concentration of nitrosamine in the medium by acidifying 5 ml carcinogen solution with 5 ml of 1% aqueous sulfuric acid and extracting the mixture with 4 ml methylene chloride. We removed the cloudiness due to suspended water by passing the organic extracts through cotton, and the nitrosamine concentration was measured by UV spectrophotometry. We detected no significant change in absorbance at 358 nm when we compared data collected be fore and after the BBN and BCPN carcinogen-medium solutions were incubated at 80±5° C for 4 days (table I). To demonstrate that none
70
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50
o
Z
W .....J
!:: 40
z
2 LU
.....J
TADLE
l.-UV absorbance data before and after the incubation of carcinogen-containing media U Carcinogen
U
Mean absorbance unitsz sn
BBN (before incubation) BBN (after incubation)
O.66±O.03 (4) O.64±O.06 (4)
BCPN (before incubation) BCPN (after incubation)
O.56±O.03 (4) O.55±O.03 (3)
U ::J
30
Z
b
20
10
Each medium was incubated without tissues for 4 days at
80±5°C. b
Numbers in parentheses are No. ofreplicates.
1
of the observed absorbance was due to a decomposition product bearing a nitrosamino function with UV properties fortuitously similar to those of the starting materials, we compared the mass spectra (solid probe, 70 eV; Jeolco 0lSG2 instrument; Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan) of the residues remaining after evaporation of the methylene chloride extracts to those of reference specimens of BBN and BCPN; except for the presence of extraneous peaks (e.g., at mle, 149) attributable to plasticizers and other contaminants introduced on incubation and work-up, the spectra of the extracts were virtually identical to those of BBN and BCPN. Finally, we checked the purity of both compounds after incubation by subjecting the methylene chloride extracts to thin-layer chromatography (Baker-flex Silica Gel IB-F plates; J. T. Baker Chemical Co., Phillipsburg, N.J.; with diethyl ether as developer and detection by fluorescence quenching); again, no evidence of decom position was detected. RESULTS
In epithelia that were fixed immediately after removal from the animal, the mean number of nuclei per unit length of epithelium was 31 (text-fig. I). This value is in elose agreement with previous measurements (6). After 7 days of culture in MB 752/1 medium without carcinogen, the mean number of nuclei per unit length of epithelium had increased to 58. Epithelia cultured in medium containing 0.5 and 2.7 mM BCPN showed an apparent increase in the mean number of nuclei per unit length over that in control cultures; however, this apparent increase was not significant as judged by Student's t-test at the 95% level. The apparent decrease in nuclei per unit length in epithelia cultured in medium
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NATL CANCER INST
2
3
4
5
6
CARCINOGEN CONCENTRATION (mM)
TEXT-FIGURE l.-Effect of BBN and BCPN on hyperplasia in bladder epithelium. Tissues were cultured continuously for 7 days in presence of BBN or BCPN. Shaded area indicates mean No. of nuclei per unit length±sE for epithelia that were not cultured but fixed immediately. All points are means z sz. _..__e, BBN; 0 - 0 , BCPN.
containing 2.9 mxt BBN was also not significant. BBN at a concentration of 5.7 mM was completely toxic to epithelial cells. None of the cultures at this concentration retained any epithelial cells after 7 days of culture; considerable damage was also evident in the submucosa. BCPN at 5.3 msr , however, did not cause any apparent loss of epithelial cells compared to uncultured controls. This concentration did appear to suppress the hyperplastic response inasmuch as the mean number of nuclei per unit length of epithelium at this concentration remained at the uncultured value. Analysis of histograms showing the frequencies of unit lengths of epithelia classified on the basis of nuclear number revealed additional information about the effects of the two carcinogens on the epithelia. Uncultured epithelia had a homogeneous population of nuclear lengths (text-fig. 2, a). This was also true with hyperplastic epithelia cultured for 7 days without carcinogen (text-fig. 2, b). Epithelia cultured in 0.5 and 2.7 mM BCPN, however, showed a more heterogeneous population of unit lengths, with most of the heterogeneity expressed as unit lengths with considerably greater numbers of nuclei than control cultures (text-fig. 2, c, d). This was also true with 0.6 and 2.9 mM BBN (textfig. 2, f, g). In addition, BBN-cultured epithelia at these concentrations showed higher frequencies of unit lengths with nuclear numbers that were lower than those of control cultures. Some of these unit lengths
VaL. 60, NO. 1, JANUARY 1978
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ACTION OF BBN AND Bep!\' ON ORGAN-CULTURED BLADDER EPITHELIUM
6
9
J
I
a
J
4
c
2 3.-0utline of epithelium cultured for 7 days in 5.3 BCPN. Arrow a indicates region of necrosis . Arrow b indicates region of hypcrplasia (see fig. I). Arrow c indicates nonhvpcrplastic region (see fig. 2). Linr at top of epithelium is one unit length (150 p.).
TEXT-FI
ABSTRACT- The effects of the bladder carcinogens butyl(4-hydroxybutyl)nitrosamine (BBN) and butyl(3-carboxypropyl)-nitrosamine (BCPN) on proliferating transitional rat epithelium in organ culture were studied. At low to intermediate concentration ranges (0.5-2.9 mM), both compounds appeared to stimulate hyperplasia in some regions of epithelia. The major effect of both carcinogens, however, was to suppress hyperplasia in other regions of epithelia and, at higher concentrations (5-6 mM), to cause necrosis. For comparableconcentrations, BBN was more effective in suppressing proliferation and causing necrosis than was BCPN. - J Natl Cancer Inst 60: 219-223, 1978.
When administered orally in the drinking water, BBN induces bladder cancer in rats (1) and mice (2). BBN is not found in the urine (3), presumably because it is metabolized in the liver. Its principal urinary metabolite has been identified as BCPN (3), which, when administered orally, has also been shown to be selectively carcinogenic to the bladder epithelium (4). Both BBN and BCPN are carcinogenic when administered by intravesicular instillation (5). Presumably the transitional epithelial cells have the ability to metabolize BBN to BCPN and, subsequently, BCPN to the ultimate carcinogen. Because little is known about the early effects and mode of action of BBN and BCPN and in view of the apparent ability of both carcinogens to act on the bladder epithelium without prior activation in other organs, studies were initiated to determine what effects BBN and BCPN might have on the bladder epithelium during early periods of exposure to these carcinogens. Since we were primarily interested in the early effects of the carcinogens on the histologic organization of the epithelium, the analysis was done in organ culture because it allows experimentation under carefully controlled and defined conditions on epithelia that are structurally intact and thus more characteristic of normal bladder epithelium than are cell cultures. By characterizing the effect of BBN and BCPN in culture, we hope to make some tentative conclusions concerning the initial effects that these carcinogens have on the epithelium in vivo. This study describes the effects of BBN and BCPN on epithelia cultured under conditions that stimulate proliferation. The effects of these carcinogens on nonproliferating transitional epithelium will be considered later.
MATERIALS AND METHODS Male 6- to 7-week-old Fischer rats were obtained VOL. 60, NO. I, JANUARY 1978
219
from the Charles River Breeding Laboratories , North Wilmington, Massachusetts. The organ-culture procedure was described in (6). In the present experiments, Waymouth's MB 752/1 medium (7) was used and cultures were maintained at 35.5° C. As in previous studies (6, 8), serum or other protein supplements were not used. Before BBN was added to the culture medium, it was dissolved in absolute ethanol. Ethanol was added to BCPN-containing medium and control medium so that all media contained the same ethanol concentrations (0.5%). All preparations of culture media were sterilized by being passed through 0.45- JL pore-size membrane filters (Millipore Corp., Bedford, Mass.). Tissues were exposed to carcinogen for the entire 7-day culture period and medium was changed at 2-day intervals. We assessed the effect of the two carcinogens on the bladder epithelium by determining the extent to which they affected the number of cells in the epithelium. We accomplished this by scoring the number of nuclei per unit length (150 JL) of epithelium from 3 or 4 organ cultures for each experimental group. This procedure was detailed in (6). BCPN (melting point, 39-40° C) was synthesized as described in (9). BBN [boiling point, 125-127° C (0.2 mm Hg)] was synthesized by Dr. EImer Reist, Stanford Research Institute, Palo Alto, California, using the method of Schmähl and Krüger (10) and was obtained from the National Cancer Institute Chemical Repository (Dr. David Longfellow, Project Officer). To demonstrate the stability of BBN and BCPN in culture, we did stability studies on both compounds, in the absence of bladder tissues, under exaggerated conABBREVIATIONS USED: BBN = butyl(4-hydroxybutyl)nitrosamine; BCPN = butyl(3-carboxypropyl)nitrosamine; m]e = mass divided by elementary charge; H & E = hematoxylin and eosin.
Received May 17, 1977; accepted August 18, 1977. Lung Cancer Branch, National Cancer Institute (NCI), National Institutes of Health, Public Health Service, V.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 Western Regional Research Center, Agricultural Research Service, V.S. Department of Agriculture, Berkeley, Calif. 94710. 4 Analytical Chemistry Section, Carcinogen Metabolism and Toxicology Branch, NCI. 5 Address reprint requests to Dr. Keefer. 6 We thank Dr. John Driscoll, Division of Cancer Treatment, NCI, for help in predicting partition coefficients, Mr. J. Richard Miller, Carcinogen Metabolism and Toxicology Branch, NCI, for the mass spectrometry measurements, and Ms. Margaret O'Boyle, Lung Cancer Branch, NCI, for typing the manuscript. 1
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REESE, FRIEDMAN, GAFFIELD, AND KEEFER
ditions of time and temperature. We determined the concentration of nitrosamine in the medium by acidifying 5 ml carcinogen solution with 5 ml of 1% aqueous sulfuric acid and extracting the mixture with 4 ml methylene chloride. We removed the cloudiness due to suspended water by passing the organic extracts through cotton, and the nitrosamine concentration was measured by UV spectrophotometry. We detected no significant change in absorbance at 358 nm when we compared data collected be fore and after the BBN and BCPN carcinogen-medium solutions were incubated at 80±5° C for 4 days (table I). To demonstrate that none
70
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50
o
Z
W .....J
!:: 40
z
2 LU
.....J
TADLE
l.-UV absorbance data before and after the incubation of carcinogen-containing media U Carcinogen
U
Mean absorbance unitsz sn
BBN (before incubation) BBN (after incubation)
O.66±O.03 (4) O.64±O.06 (4)
BCPN (before incubation) BCPN (after incubation)
O.56±O.03 (4) O.55±O.03 (3)
U ::J
30
Z
b
20
10
Each medium was incubated without tissues for 4 days at
80±5°C. b
Numbers in parentheses are No. ofreplicates.
1
of the observed absorbance was due to a decomposition product bearing a nitrosamino function with UV properties fortuitously similar to those of the starting materials, we compared the mass spectra (solid probe, 70 eV; Jeolco 0lSG2 instrument; Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan) of the residues remaining after evaporation of the methylene chloride extracts to those of reference specimens of BBN and BCPN; except for the presence of extraneous peaks (e.g., at mle, 149) attributable to plasticizers and other contaminants introduced on incubation and work-up, the spectra of the extracts were virtually identical to those of BBN and BCPN. Finally, we checked the purity of both compounds after incubation by subjecting the methylene chloride extracts to thin-layer chromatography (Baker-flex Silica Gel IB-F plates; J. T. Baker Chemical Co., Phillipsburg, N.J.; with diethyl ether as developer and detection by fluorescence quenching); again, no evidence of decom position was detected. RESULTS
In epithelia that were fixed immediately after removal from the animal, the mean number of nuclei per unit length of epithelium was 31 (text-fig. I). This value is in elose agreement with previous measurements (6). After 7 days of culture in MB 752/1 medium without carcinogen, the mean number of nuclei per unit length of epithelium had increased to 58. Epithelia cultured in medium containing 0.5 and 2.7 mM BCPN showed an apparent increase in the mean number of nuclei per unit length over that in control cultures; however, this apparent increase was not significant as judged by Student's t-test at the 95% level. The apparent decrease in nuclei per unit length in epithelia cultured in medium
J
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2
3
4
5
6
CARCINOGEN CONCENTRATION (mM)
TEXT-FIGURE l.-Effect of BBN and BCPN on hyperplasia in bladder epithelium. Tissues were cultured continuously for 7 days in presence of BBN or BCPN. Shaded area indicates mean No. of nuclei per unit length±sE for epithelia that were not cultured but fixed immediately. All points are means z sz. _..__e, BBN; 0 - 0 , BCPN.
containing 2.9 mxt BBN was also not significant. BBN at a concentration of 5.7 mM was completely toxic to epithelial cells. None of the cultures at this concentration retained any epithelial cells after 7 days of culture; considerable damage was also evident in the submucosa. BCPN at 5.3 msr , however, did not cause any apparent loss of epithelial cells compared to uncultured controls. This concentration did appear to suppress the hyperplastic response inasmuch as the mean number of nuclei per unit length of epithelium at this concentration remained at the uncultured value. Analysis of histograms showing the frequencies of unit lengths of epithelia classified on the basis of nuclear number revealed additional information about the effects of the two carcinogens on the epithelia. Uncultured epithelia had a homogeneous population of nuclear lengths (text-fig. 2, a). This was also true with hyperplastic epithelia cultured for 7 days without carcinogen (text-fig. 2, b). Epithelia cultured in 0.5 and 2.7 mM BCPN, however, showed a more heterogeneous population of unit lengths, with most of the heterogeneity expressed as unit lengths with considerably greater numbers of nuclei than control cultures (text-fig. 2, c, d). This was also true with 0.6 and 2.9 mM BBN (textfig. 2, f, g). In addition, BBN-cultured epithelia at these concentrations showed higher frequencies of unit lengths with nuclear numbers that were lower than those of control cultures. Some of these unit lengths
VaL. 60, NO. 1, JANUARY 1978
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ACTION OF BBN AND Bep!\' ON ORGAN-CULTURED BLADDER EPITHELIUM
6
9
J
I
a
J
4
c
2 3.-0utline of epithelium cultured for 7 days in 5.3 BCPN. Arrow a indicates region of necrosis . Arrow b indicates region of hypcrplasia (see fig. I). Arrow c indicates nonhvpcrplastic region (see fig. 2). Linr at top of epithelium is one unit length (150 p.).
TEXT-FI
