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The effect of dimethylbenzanthracene on the incorporation of [3H] thymidine into DNA of rat mammary gland and uterus
Europ. 07. Cancer Vol. 6, pp. t 11-113. Pergamon Press 1970. Printed in Great Britain
The Effect of Dimethylbenzanthracene on the Incorporation of Thymidine into DNA of Rat Mammary Gland and Uterus R. J. B. KING and DOROTHEA M. COWAN Departmentsof HormoneBiochemistryand Pathology, Imperial CancerResearchFund, Lincoln's Inn Fields, London W.C.2., England
tube 24 hr before the [3H] thymidine. Oestradiol injections were given in 0.2 ml corn oil s.c. in a mid-dorsal position between glands 3 and 4.
INTRODUCTION THE CARCINOGENIC effect of dimethylbcnzanthracene (DMBA) on rat m a m m a r y gland is well documented [1] but the mechanism by which this occurs is unknown. DMBA prolongs the S phase of the cell cycle of mouse hair follicoles [2] and other authors have reported inhibitory effects on DNA synthesis [3-5]. In view of this it was of interest to study the effect of DMBA on DNA synthesis in the m a m m a r y gland. As ovarian hormones influence tumorigenesis in the mammary gland [1], the effect of these on DNA synthesis has also been studied.
Removal of mammary glands The whole pelt was removed and fixed in Bouin's fluid for 24 hr to visualise the glands. The majority of experiments were carried out on tissue from the centre of the gland which consisted of ducts and side buds. In experiments where only the terminal buds were used, only the large club shaped buds at the periphery of the gland, opposite the nipple, were used. It was sometimes necessary to combine buds from more than one gland to obtain sufficient material.
METHODS Animals Virgin female Sprague-Dawley rats were acclimatised to a cycle of 12 hr light and 12 hr dark with handling 2-3 hr after the beginning of the light period.
Oestrus cycle This was determined by daily vaginal smears taken 3 hr after the beginning of the light period. This was continued for 1-2 weeks before the rats were used.
Administration of chemicals In all cases, these were given 3 hr after the beginning of the light period. [SH] thymidine (s.a. 3-5C/mmole, Radiochemical Centre, Amersham) was injected (intra peritoneal 1 ~tc/g. body weight) on the 50th day of life, 22 hr before death. DMBA or benzanthracene (30 mg in 2 ml corn oil) were given by stomach
Extraction of DWA The mammary gland was weighed, minced with scissors and then disrupted with a Silverson (Silverson Limited, London) homogeniser in 5 ml water. Ten ml of N perchloric acid (PCA) was added and the suspension centrifuged. The pellet was washed three times each with 10 ml of 0.5 N PCA followed by three washings each of 10 ml ethanol: ether: chloroform (2 : 2 : 1).
Submitted for publication 7 July 1969. Accepted 20 October 1969.
111
112
R. 07. B. King and Dorothea M. Cowan Table 1. Incorporationof [sH] thymidine into rat mammary gland and uterus DPM/gtg DNA
D P M / m g weight
gtg DNA/g weight
Mammary gland
Uterus
Mammary gland
2324-86
884-13
1184-25
2004-31
5884-45
22844-117
Oestrus + DMBA
354-8
454-4
13-4-3
1224-33
6674-98
26114-569
Oestrus+ benzanthracene
444-11
--
314-11
--
7534-117
Prooestrus
444-13 3284-163
474-25
8974-535
8234-184
24464-223 16154-127
Oestrus
Prooestxns + DMBA
Uterus
Mammary gland
94-2
1194-49
64-2
1974-77
9944- 159
Ovariectomized
774-25
--
101 4-52
--
7594-138
Ovariectomized+ 1 lag oestradiol
954-22
--
102±21
--
11604-243
5654-1 I0
--
24464-1354
714-27
--
13984-62
Ovariectomized-{3 x I0 lag oestradlol* 2804-46 Ovariectomized + 3 × 10 gtg oestradiol+ DMBA
504-17
Uterus
--
--
Results are expressed as m e a n + S . E . M , for at least 4 estimations. *Only 3 experiments were carried out with this group.
This removed all of the acid-soluble tritium. The insoluble residue was heated for 15 min. at 70 ° with 1.5 ml 0.5 N PCA and 0.5 aliquots of the soluble fraction taken for DNA [6] and tritium estimations. The tritium was counted in a Packard liquid scintillation spectrometer model 3003 and quenching was assessed by channel ratio [7]. In six experiments, [SH] thymidine labelled m a m m a r y glands were removed without fixation in Bouin's fluid and the D N A extracted as above. The specific activity of the DNA (disintegrations/min/~tg DNA) was the same as in the fixed samples, indicating that fixation does not alter this parameter. RESULTS
Effect of oestrous cycle and hydrocarbons on thymidine incorporation In the m a m m a r y gland, [SH] thymidine incorporation per ~tg DNA was higher at oestrus than at prooestrus (p<0.001) whilst the converse was true of the uterus ( p < 0 . 0 5 ) , Table 1. In both tissues, there was a higher mean DNA content at prooestrus than at oestrus but this was not statistically significant ( p > 0 . 0 5 ) . Ovariectomy 7 days prior to [SH] thymidine injection lowered the incorporation in the m a m m a r y gland relative to the oestrus level. A single injection of 1 lag oestradiol 24 hr prior
to the [SH] thymidine did not affect [SH] thymidine incorporation but three daily injections, each of 10 rtg, increased incorporation to the oestrus level. Administration of DMBA 24 hr prior to the [SH] thymidine markedly inhibited both the prooestrus and oestrus levels of incorporation in m a m m a r y gland ( p < 0 . 0 2 and 0.001 respectively). The non-carcinogenic hydrocarbon benzanthracene had a similar effect at oestrus but was not tested at prooestrus. DMBA also inhibited the effect of oestradiol on thymidine incorporation.
Thymidine incorporation into the terminal buds and central area of the mammary gland In six experiments, the incorporation was compared between the central area and terminal buds from the same gland. As the terminal buds contained too little DNA to measure, this comparison was made per g wet weight. Both at prooestrus and oestrus there was a greater incorporation into the terminal buds than into the central region and this difference was greater at prooestrus than at oestrus. The ratio of incorporation into the terminal buds: central area of the same gland was 1 0 : 2 at prooestrus and 3 : 2 at oestrus. DMBA inhibited [SH] thymidine incorporation into the terminal buds. All of the above analyses were carried out
The Effect of Dimethylbenzanthracene on Rat Mammary Gland and Uterus on m a m m a r y tissue from gland 3. Incorporation into the central areas of the other glands occurred to about the same extent except that there was a tendency for a lower incorporation into gland 6.
DISCUSSION The observation that maximal [SH] thymidine incorporation occurred at oestrus in the m a m m a r y gland and at prooestrus in the uterus indicates that these two tissues have different sensitivities to endogenous hormones. Hydrocarbon inhibition of DNA synthesis has been reported for a number of tissues both in vivo and in vitro [3-5] but the mechanism is unknown. In the present experiments, it is possible that the hydrocarbon is affecting DNA
113
synthesis indirectly via the endocrine glands. The lack of effect on R N A synthesis [8] would indicate that it is not simply a general toxic effect of the hydrocarbon. Since inhibition occurs in tissues in which the hydrocarbon is not carcinogenic and, in the present experimenu, the non-carcinogenic hydrocarbon, benzanthracene, was also inhibitory, it is unlikely that it is directly related to carcinogenesis. However, it may contribute to some, as yet undefined, carcinogenic effect of the hydrocarbon. Although the elevation of DNA in the DMBA treated animals was not statistically significant it is consistent with the report of Alfred [8] that DMBA increased cellular DNA, and also suggests that this hydrocarbon may affect DNA breakdown.
SUMMARY The in vivo incorporation of [3H] thymidine into rat mammary gland has been studied under different conditions. Incorporation into D,N'A was higher in glands obtained at oestrus than at prooestrus and decreased after ovariectomy. The effect of ovariectomy was prevented by oestrudiol injection. The incorporation of [~H] thymidine was greater in the terminal buds of the mammary gland than in the central area of the gland. D M B A markedly inhibited incorporation into DNA of glands obtained at both oestrus and prooestrus. It also inhibited the incorporation into uterine DNA. The non-carcinogenic hydrocarbon, benzanthracene, also inhibited D N A synthesis in mammary gland. It is concluded that the D M B A inhibition of D N A synthesis is not directly related to the carcinogenic action of this hydrocarbon.
REFERENCES 1. T . L . DAO, Studies on the mechanism of carcinogenesis in the mammary gland. Progr. exp. Tumor Res. 11, 235 (1961). 2. J . A . McCARTER and H. Q.UASTEL,Note on the effect of a carcinogenic hydrocarbon on the synthesis of deoxyribonucleic acid. Biochim. biophys. Acta 55, 553 (1962). 3. J . A . McCARTER and H. Q UASTET.,The effect of dimethylbenzanthracene on the cellular proliferation cycle. Nature (Lond.) 194, 873 (1962). 4. E.V. J~.NSEN, E. FORD and C. HUGOINS,Depressed incorporation of thymidine3H into deoxyribonucleic acid following administration of 7, 12, dimethylbenz (a)anthracene. Proc. nat. Acad. Sci. 50, 454 (1963). 5. L.J. ALFRED and V. HANIFIN, Suppression of "early" DNA synthesis in hydrocarbon carcinogen treated cultures. Proc. Amer. Ass. Cancer Res. 7, 2 (1966). 6. K. BURTON, A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. 07. 62, 315 (1956). 7. L . A . BAILT.IE,Determination of liquid scintillation counting by pulse height shift. Int. 07. appl. Radiat. Isotopes 8, 1 (1960). 8. L.J. ALFRED, Differential inhibition of deoxyribonucleic acid and ribonucleic acid synthesis induced in cultured mammalian cells by 7, 12 dimethylbenzanthracene. Nature (Lond.) 208, 1339 (1965).
The Effect of Dimethylbenzanthracene on the Incorporation of Thymidine into DNA of Rat Mammary Gland and Uterus R. J. B. KING and DOROTHEA M. COWAN Departmentsof HormoneBiochemistryand Pathology, Imperial CancerResearchFund, Lincoln's Inn Fields, London W.C.2., England
tube 24 hr before the [3H] thymidine. Oestradiol injections were given in 0.2 ml corn oil s.c. in a mid-dorsal position between glands 3 and 4.
INTRODUCTION THE CARCINOGENIC effect of dimethylbcnzanthracene (DMBA) on rat m a m m a r y gland is well documented [1] but the mechanism by which this occurs is unknown. DMBA prolongs the S phase of the cell cycle of mouse hair follicoles [2] and other authors have reported inhibitory effects on DNA synthesis [3-5]. In view of this it was of interest to study the effect of DMBA on DNA synthesis in the m a m m a r y gland. As ovarian hormones influence tumorigenesis in the mammary gland [1], the effect of these on DNA synthesis has also been studied.
Removal of mammary glands The whole pelt was removed and fixed in Bouin's fluid for 24 hr to visualise the glands. The majority of experiments were carried out on tissue from the centre of the gland which consisted of ducts and side buds. In experiments where only the terminal buds were used, only the large club shaped buds at the periphery of the gland, opposite the nipple, were used. It was sometimes necessary to combine buds from more than one gland to obtain sufficient material.
METHODS Animals Virgin female Sprague-Dawley rats were acclimatised to a cycle of 12 hr light and 12 hr dark with handling 2-3 hr after the beginning of the light period.
Oestrus cycle This was determined by daily vaginal smears taken 3 hr after the beginning of the light period. This was continued for 1-2 weeks before the rats were used.
Administration of chemicals In all cases, these were given 3 hr after the beginning of the light period. [SH] thymidine (s.a. 3-5C/mmole, Radiochemical Centre, Amersham) was injected (intra peritoneal 1 ~tc/g. body weight) on the 50th day of life, 22 hr before death. DMBA or benzanthracene (30 mg in 2 ml corn oil) were given by stomach
Extraction of DWA The mammary gland was weighed, minced with scissors and then disrupted with a Silverson (Silverson Limited, London) homogeniser in 5 ml water. Ten ml of N perchloric acid (PCA) was added and the suspension centrifuged. The pellet was washed three times each with 10 ml of 0.5 N PCA followed by three washings each of 10 ml ethanol: ether: chloroform (2 : 2 : 1).
Submitted for publication 7 July 1969. Accepted 20 October 1969.
111
112
R. 07. B. King and Dorothea M. Cowan Table 1. Incorporationof [sH] thymidine into rat mammary gland and uterus DPM/gtg DNA
D P M / m g weight
gtg DNA/g weight
Mammary gland
Uterus
Mammary gland
2324-86
884-13
1184-25
2004-31
5884-45
22844-117
Oestrus + DMBA
354-8
454-4
13-4-3
1224-33
6674-98
26114-569
Oestrus+ benzanthracene
444-11
--
314-11
--
7534-117
Prooestrus
444-13 3284-163
474-25
8974-535
8234-184
24464-223 16154-127
Oestrus
Prooestxns + DMBA
Uterus
Mammary gland
94-2
1194-49
64-2
1974-77
9944- 159
Ovariectomized
774-25
--
101 4-52
--
7594-138
Ovariectomized+ 1 lag oestradiol
954-22
--
102±21
--
11604-243
5654-1 I0
--
24464-1354
714-27
--
13984-62
Ovariectomized-{3 x I0 lag oestradlol* 2804-46 Ovariectomized + 3 × 10 gtg oestradiol+ DMBA
504-17
Uterus
--
--
Results are expressed as m e a n + S . E . M , for at least 4 estimations. *Only 3 experiments were carried out with this group.
This removed all of the acid-soluble tritium. The insoluble residue was heated for 15 min. at 70 ° with 1.5 ml 0.5 N PCA and 0.5 aliquots of the soluble fraction taken for DNA [6] and tritium estimations. The tritium was counted in a Packard liquid scintillation spectrometer model 3003 and quenching was assessed by channel ratio [7]. In six experiments, [SH] thymidine labelled m a m m a r y glands were removed without fixation in Bouin's fluid and the D N A extracted as above. The specific activity of the DNA (disintegrations/min/~tg DNA) was the same as in the fixed samples, indicating that fixation does not alter this parameter. RESULTS
Effect of oestrous cycle and hydrocarbons on thymidine incorporation In the m a m m a r y gland, [SH] thymidine incorporation per ~tg DNA was higher at oestrus than at prooestrus (p<0.001) whilst the converse was true of the uterus ( p < 0 . 0 5 ) , Table 1. In both tissues, there was a higher mean DNA content at prooestrus than at oestrus but this was not statistically significant ( p > 0 . 0 5 ) . Ovariectomy 7 days prior to [SH] thymidine injection lowered the incorporation in the m a m m a r y gland relative to the oestrus level. A single injection of 1 lag oestradiol 24 hr prior
to the [SH] thymidine did not affect [SH] thymidine incorporation but three daily injections, each of 10 rtg, increased incorporation to the oestrus level. Administration of DMBA 24 hr prior to the [SH] thymidine markedly inhibited both the prooestrus and oestrus levels of incorporation in m a m m a r y gland ( p < 0 . 0 2 and 0.001 respectively). The non-carcinogenic hydrocarbon benzanthracene had a similar effect at oestrus but was not tested at prooestrus. DMBA also inhibited the effect of oestradiol on thymidine incorporation.
Thymidine incorporation into the terminal buds and central area of the mammary gland In six experiments, the incorporation was compared between the central area and terminal buds from the same gland. As the terminal buds contained too little DNA to measure, this comparison was made per g wet weight. Both at prooestrus and oestrus there was a greater incorporation into the terminal buds than into the central region and this difference was greater at prooestrus than at oestrus. The ratio of incorporation into the terminal buds: central area of the same gland was 1 0 : 2 at prooestrus and 3 : 2 at oestrus. DMBA inhibited [SH] thymidine incorporation into the terminal buds. All of the above analyses were carried out
The Effect of Dimethylbenzanthracene on Rat Mammary Gland and Uterus on m a m m a r y tissue from gland 3. Incorporation into the central areas of the other glands occurred to about the same extent except that there was a tendency for a lower incorporation into gland 6.
DISCUSSION The observation that maximal [SH] thymidine incorporation occurred at oestrus in the m a m m a r y gland and at prooestrus in the uterus indicates that these two tissues have different sensitivities to endogenous hormones. Hydrocarbon inhibition of DNA synthesis has been reported for a number of tissues both in vivo and in vitro [3-5] but the mechanism is unknown. In the present experiments, it is possible that the hydrocarbon is affecting DNA
113
synthesis indirectly via the endocrine glands. The lack of effect on R N A synthesis [8] would indicate that it is not simply a general toxic effect of the hydrocarbon. Since inhibition occurs in tissues in which the hydrocarbon is not carcinogenic and, in the present experimenu, the non-carcinogenic hydrocarbon, benzanthracene, was also inhibitory, it is unlikely that it is directly related to carcinogenesis. However, it may contribute to some, as yet undefined, carcinogenic effect of the hydrocarbon. Although the elevation of DNA in the DMBA treated animals was not statistically significant it is consistent with the report of Alfred [8] that DMBA increased cellular DNA, and also suggests that this hydrocarbon may affect DNA breakdown.
SUMMARY The in vivo incorporation of [3H] thymidine into rat mammary gland has been studied under different conditions. Incorporation into D,N'A was higher in glands obtained at oestrus than at prooestrus and decreased after ovariectomy. The effect of ovariectomy was prevented by oestrudiol injection. The incorporation of [~H] thymidine was greater in the terminal buds of the mammary gland than in the central area of the gland. D M B A markedly inhibited incorporation into DNA of glands obtained at both oestrus and prooestrus. It also inhibited the incorporation into uterine DNA. The non-carcinogenic hydrocarbon, benzanthracene, also inhibited D N A synthesis in mammary gland. It is concluded that the D M B A inhibition of D N A synthesis is not directly related to the carcinogenic action of this hydrocarbon.
REFERENCES 1. T . L . DAO, Studies on the mechanism of carcinogenesis in the mammary gland. Progr. exp. Tumor Res. 11, 235 (1961). 2. J . A . McCARTER and H. Q.UASTEL,Note on the effect of a carcinogenic hydrocarbon on the synthesis of deoxyribonucleic acid. Biochim. biophys. Acta 55, 553 (1962). 3. J . A . McCARTER and H. Q UASTET.,The effect of dimethylbenzanthracene on the cellular proliferation cycle. Nature (Lond.) 194, 873 (1962). 4. E.V. J~.NSEN, E. FORD and C. HUGOINS,Depressed incorporation of thymidine3H into deoxyribonucleic acid following administration of 7, 12, dimethylbenz (a)anthracene. Proc. nat. Acad. Sci. 50, 454 (1963). 5. L.J. ALFRED and V. HANIFIN, Suppression of "early" DNA synthesis in hydrocarbon carcinogen treated cultures. Proc. Amer. Ass. Cancer Res. 7, 2 (1966). 6. K. BURTON, A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. 07. 62, 315 (1956). 7. L . A . BAILT.IE,Determination of liquid scintillation counting by pulse height shift. Int. 07. appl. Radiat. Isotopes 8, 1 (1960). 8. L.J. ALFRED, Differential inhibition of deoxyribonucleic acid and ribonucleic acid synthesis induced in cultured mammalian cells by 7, 12 dimethylbenzanthracene. Nature (Lond.) 208, 1339 (1965).