Znt. J. Cancer: 20, 292-295 (1977)
POSSIBLE TWO-STAGE TRANSPLACENTAL LIVER CARCINOGENESIS IN C57BL/6 MICE Vlasta ARMUTH and Isaac BERENBLUM Experimental Biorogy Unit, Weizmann Znstitute of Science, Rehovot, Israel
A single SC injection of 2-acetylaminofluorene (AAF) was given to pregnant C57BLl6 mice on day 15 of gestation, and the offspring subsequently given twice-weekly injections of phorbol for 25 weeks. Control groups included: (I)untreated; (2) AAFtreated mothers (kept under observation for f8 months, as with all the other groups); (3) untreated offspring of untreated mothers; (4) untreated offspring of AAF-treated mothers, and (5) phorboltreated offspring of untreated mothers. The incidence of hepatomas in the phorbol-treated offspring of AAF-injected mothers was 8/74 (ll'?'~), as corn. pared with 2/80 (2.5%) in the untreated offspring of AAF-injected mothers. The AAF-injected mothers themselves developed 3/36 (8%) hepatomas; while all the other control groups were free from liver tumours. The development of reticulum cell sarcomas, and of a few cases of lung adenomas, in the various groups, was presumably spontaneous. The results seem sufficiently encouraging, as a model for the study of systemic carcinogenesis, to warrant further attempts a t two-stage transplacental carcinogenesis, using other potential initiators and promoters.
Wattenberg (1970), who injected benzo(a)pyrene SC into pregnant ICR mice, and later applied croton oil to the skin of the offspring. In addition to papillomas at the site of the croton oil applications, there appeared to be an increase in lung adenomas in the offspring. MATERIAL AND METHODS
Three-month-old male and female C57BL/6 mice, from the Institute's Breeding Centre, were kept together overnight, and the females checked the next morning for vaginal plugs. That day was counted as day 0 of gestation, the females having been examined again 2 weeks later for visible signs of pregnancy. On day 15 of pregnancy, 0.5 ml of a 2 % solution of 2-acetylaminofluorene (AAF) in tricaprylin, was injected SC to half of the pregnant mice. Following delivery, which took place on days 18-20, half of the young were given I P injections Exposure of pregnant animals to chemical carci- of 0.25 pmole of phorbol in phosphate-buffered nogens can lead to tumour development in the saline, twice weekly, from the age of 10 days; offspring, provided the carcinogen, or its active after weaning, at the age of 3 weeks, the dose of metabolite, is able to pass through the placenta phorbol was doubled to 0.5 pmole per injection. without being destroyed in the process (Napalkov, The phorbol treatment was continued for 25 weeks 1973). Such transplacental carcinogenesis should in half the offspring of the AAF-treated mothers, lend itself ideally to the study of the mechanism and also in half the offspring of untreated mothers. of systemic carcinogenesis, by the adaptation of The young were separated, after weaning, accordthe two-stage technique, i.e. by administering the ing to sex and form of treatment, and were housed, initiator to the pregnant mother and the promoter 5-6 per metal cage, in an air-conditioned room at to the offspring after birth. 20-24°C. They received a well-balanced diet, in As a first attempt, in a series of planned exper- pellet form, produced at the Institute, and tap iments, to explore the possibilities and scope of water ad libitum. The mothers were also kept under two-stage transplacental carcinogenesis, phorbolobservation throughout the experiment. the unesterified parent alcohol of croton oil's The experimental groups were made up as follows: active principle, TPA (Hecker, 1968)-was chosen (1) pregnant mothers, injected with AAF on day 15 for postnatal promoting action, and administered to the offspring of C57BL/6 mothers that had received of pregnancy; (2) untreated pregnant mothers; a single SC injection of 2-acetylaminofluorene (3) male and female offspring of untreated mothers, (AAF) as initiator. Phorbol had previously been which received no treatment, i.e. untreated controls; (4) male and female offspring of untreated mothers, found to act as a promoting agent for lung and receiving phorbol injections for 25 weeks; ( 5 ) male liver carcinogenesis in mice (Armuth and Berenblum, and female offspring of AAF-treated mothers, 1972) and for mammary carcinogenesis in rats (Armuth and Berenblum, 1974). The only previous attempt at two-stage transReceived: March 16, 1977 and in revised form May 12, placental carcinogenesis was made by Bulay and 1977.
293
TWO-STAGE TRANSPLACENTAL CARCINOGENESIS TABLE I TUMOUR INCIDENCE I N ADULT C57BL/6 MICE INJECTED (DURING PREGNANCY) SUBCUTANEOUSLY WITH 2-ACETYLAMINOFLUORENE ~~
Number of mice at start of experiment
Treatment ~~
~~
Tumour incidence per effective total
a
Hepatomas
Reticulum cell sarcomas
Other tumours
48
3/36 = 8% (586 d) 0/28 = 0%
2/36 = 5% (586 d) 0/28= 0%
1 with lung adenoma
29
~
AAF a to pregnant mice Untreated (pregnant) mice
-
' Effective total = survivors at the time of the first tumour detected in the experiment. - Number in parenthesis animals at the time the tumours were detected. - a Single SC injection of 0.5 ml of 2 % AAF in tricaprylin.
without further treatment; (6) male and female offspring of AAF-treated mothers, receiving phorbol injections for 25 weeks. The animals were checked daily, and examined more thoroughly twice weekly. Moribund mice, and those with palpable tumours, were killed and autopsied, their organs fixed in Bouin's solution and embedded in paraffin, after which histological sections were stained with haematoxylin and eosin. Animals found dead in their cages were similarly autopsied, as well as all survivors at the age of 18 months, when the experiment was terminated. RESULTS
All the treatments were well tolerated by the animals, and no early deaths were recorded in any of the experimental groups. In a few of the AAFinjected mothers, the skin became depilated at the site of the SC injection, and in one case became ulcerated ;but these lesions all healed spontaneously.
=
average age of
The results, tumour incidences and average latent periods, are summarized in Tables I and 11. The pregnant mothers, injected once only with AAF, developed hepatomas in 3/36 cases and reticulum cell neoplasms in 2, with 1 case of lung adenoma (see Table I). The hepatomas are presumably attributable to the AAF treatment (cf Kirby, 1948, using SC injections of 2-aminofluorene, in C57BL mice). The reticulum cell sarcomas can be discounted as being of spontaneous origin, since they have been repeatedly found in our colony of aging C57BL mice. Transplacentally, AAF alone (Le. without phorbol treatment to the offspring) did not significantly raise the tumour incidence; nor did phorbol treatment alone (in offspring of untreated mothers) show an increase in tumour incidence (Table 11). When, however, continued phorbol treatment was given to the offspring of mothers that had received an injection of AAF, the incidence of hepatomas
TABLE I1 TUMOUR INCIDENCE IN PHORBOL-TREATED OFFSPRING OF C57BL!6 FEMALES INJECTED WITH AAF DURING PREGNANCY Treatment Sex
No. of mice at start of experiment
To mother
To offspring
-
-
F
29
-
M
-
F M
Phorbol Phorbol Phorbol Phorbol
F M
40 45 41 9 15 49 37
AAF' AAF AAF AAF
F M
Tumour incidence per effective total Hepatomas
0128 0133 1/42= 2 % (450) 1/38 = 2.5 % (450) 0/9 011 5 5/42= 12% (475) 3/32 = 9 % (475)
Reticulum cell sarcomas
' Other turnours
0128 3/33 = 9% (507)* 6/42= 14% (450) 1/38 = 2.5 % (450) One with lung adenoma (450) 019 One with lung adenoma (406) 0115 One with sarcoma SC (406) 5/42= 12% (466) 0132 -
' Effective total=number of survivors in the group at the time of appearance of first turnour. - Number in parenthesis=average age of animals bearing turnours (in days). - a AAF = 0.5 ml of 2 % acetylaminofluorene SC on day I 5 of gestation. - ' 0.25 rmole of phorbol in phosphate-buffered saline IP, twice weekly, till weaning; then 0.5 @moleIP, twice weekly, for a total of 25 weeks; (cf Armuth, 1976, for similarly negative results with phorbol alone in C57BL/6 mice).
294
ARMUTH AND BERENBLUM
was definitely raised (8/74 = 11 %, as against 2/80 = 2.5% for males plus females: - significance : 0.02 < p
POSSIBLE TWO-STAGE TRANSPLACENTAL LIVER CARCINOGENESIS IN C57BL/6 MICE Vlasta ARMUTH and Isaac BERENBLUM Experimental Biorogy Unit, Weizmann Znstitute of Science, Rehovot, Israel
A single SC injection of 2-acetylaminofluorene (AAF) was given to pregnant C57BLl6 mice on day 15 of gestation, and the offspring subsequently given twice-weekly injections of phorbol for 25 weeks. Control groups included: (I)untreated; (2) AAFtreated mothers (kept under observation for f8 months, as with all the other groups); (3) untreated offspring of untreated mothers; (4) untreated offspring of AAF-treated mothers, and (5) phorboltreated offspring of untreated mothers. The incidence of hepatomas in the phorbol-treated offspring of AAF-injected mothers was 8/74 (ll'?'~), as corn. pared with 2/80 (2.5%) in the untreated offspring of AAF-injected mothers. The AAF-injected mothers themselves developed 3/36 (8%) hepatomas; while all the other control groups were free from liver tumours. The development of reticulum cell sarcomas, and of a few cases of lung adenomas, in the various groups, was presumably spontaneous. The results seem sufficiently encouraging, as a model for the study of systemic carcinogenesis, to warrant further attempts a t two-stage transplacental carcinogenesis, using other potential initiators and promoters.
Wattenberg (1970), who injected benzo(a)pyrene SC into pregnant ICR mice, and later applied croton oil to the skin of the offspring. In addition to papillomas at the site of the croton oil applications, there appeared to be an increase in lung adenomas in the offspring. MATERIAL AND METHODS
Three-month-old male and female C57BL/6 mice, from the Institute's Breeding Centre, were kept together overnight, and the females checked the next morning for vaginal plugs. That day was counted as day 0 of gestation, the females having been examined again 2 weeks later for visible signs of pregnancy. On day 15 of pregnancy, 0.5 ml of a 2 % solution of 2-acetylaminofluorene (AAF) in tricaprylin, was injected SC to half of the pregnant mice. Following delivery, which took place on days 18-20, half of the young were given I P injections Exposure of pregnant animals to chemical carci- of 0.25 pmole of phorbol in phosphate-buffered nogens can lead to tumour development in the saline, twice weekly, from the age of 10 days; offspring, provided the carcinogen, or its active after weaning, at the age of 3 weeks, the dose of metabolite, is able to pass through the placenta phorbol was doubled to 0.5 pmole per injection. without being destroyed in the process (Napalkov, The phorbol treatment was continued for 25 weeks 1973). Such transplacental carcinogenesis should in half the offspring of the AAF-treated mothers, lend itself ideally to the study of the mechanism and also in half the offspring of untreated mothers. of systemic carcinogenesis, by the adaptation of The young were separated, after weaning, accordthe two-stage technique, i.e. by administering the ing to sex and form of treatment, and were housed, initiator to the pregnant mother and the promoter 5-6 per metal cage, in an air-conditioned room at to the offspring after birth. 20-24°C. They received a well-balanced diet, in As a first attempt, in a series of planned exper- pellet form, produced at the Institute, and tap iments, to explore the possibilities and scope of water ad libitum. The mothers were also kept under two-stage transplacental carcinogenesis, phorbolobservation throughout the experiment. the unesterified parent alcohol of croton oil's The experimental groups were made up as follows: active principle, TPA (Hecker, 1968)-was chosen (1) pregnant mothers, injected with AAF on day 15 for postnatal promoting action, and administered to the offspring of C57BL/6 mothers that had received of pregnancy; (2) untreated pregnant mothers; a single SC injection of 2-acetylaminofluorene (3) male and female offspring of untreated mothers, (AAF) as initiator. Phorbol had previously been which received no treatment, i.e. untreated controls; (4) male and female offspring of untreated mothers, found to act as a promoting agent for lung and receiving phorbol injections for 25 weeks; ( 5 ) male liver carcinogenesis in mice (Armuth and Berenblum, and female offspring of AAF-treated mothers, 1972) and for mammary carcinogenesis in rats (Armuth and Berenblum, 1974). The only previous attempt at two-stage transReceived: March 16, 1977 and in revised form May 12, placental carcinogenesis was made by Bulay and 1977.
293
TWO-STAGE TRANSPLACENTAL CARCINOGENESIS TABLE I TUMOUR INCIDENCE I N ADULT C57BL/6 MICE INJECTED (DURING PREGNANCY) SUBCUTANEOUSLY WITH 2-ACETYLAMINOFLUORENE ~~
Number of mice at start of experiment
Treatment ~~
~~
Tumour incidence per effective total
a
Hepatomas
Reticulum cell sarcomas
Other tumours
48
3/36 = 8% (586 d) 0/28 = 0%
2/36 = 5% (586 d) 0/28= 0%
1 with lung adenoma
29
~
AAF a to pregnant mice Untreated (pregnant) mice
-
' Effective total = survivors at the time of the first tumour detected in the experiment. - Number in parenthesis animals at the time the tumours were detected. - a Single SC injection of 0.5 ml of 2 % AAF in tricaprylin.
without further treatment; (6) male and female offspring of AAF-treated mothers, receiving phorbol injections for 25 weeks. The animals were checked daily, and examined more thoroughly twice weekly. Moribund mice, and those with palpable tumours, were killed and autopsied, their organs fixed in Bouin's solution and embedded in paraffin, after which histological sections were stained with haematoxylin and eosin. Animals found dead in their cages were similarly autopsied, as well as all survivors at the age of 18 months, when the experiment was terminated. RESULTS
All the treatments were well tolerated by the animals, and no early deaths were recorded in any of the experimental groups. In a few of the AAFinjected mothers, the skin became depilated at the site of the SC injection, and in one case became ulcerated ;but these lesions all healed spontaneously.
=
average age of
The results, tumour incidences and average latent periods, are summarized in Tables I and 11. The pregnant mothers, injected once only with AAF, developed hepatomas in 3/36 cases and reticulum cell neoplasms in 2, with 1 case of lung adenoma (see Table I). The hepatomas are presumably attributable to the AAF treatment (cf Kirby, 1948, using SC injections of 2-aminofluorene, in C57BL mice). The reticulum cell sarcomas can be discounted as being of spontaneous origin, since they have been repeatedly found in our colony of aging C57BL mice. Transplacentally, AAF alone (Le. without phorbol treatment to the offspring) did not significantly raise the tumour incidence; nor did phorbol treatment alone (in offspring of untreated mothers) show an increase in tumour incidence (Table 11). When, however, continued phorbol treatment was given to the offspring of mothers that had received an injection of AAF, the incidence of hepatomas
TABLE I1 TUMOUR INCIDENCE IN PHORBOL-TREATED OFFSPRING OF C57BL!6 FEMALES INJECTED WITH AAF DURING PREGNANCY Treatment Sex
No. of mice at start of experiment
To mother
To offspring
-
-
F
29
-
M
-
F M
Phorbol Phorbol Phorbol Phorbol
F M
40 45 41 9 15 49 37
AAF' AAF AAF AAF
F M
Tumour incidence per effective total Hepatomas
0128 0133 1/42= 2 % (450) 1/38 = 2.5 % (450) 0/9 011 5 5/42= 12% (475) 3/32 = 9 % (475)
Reticulum cell sarcomas
' Other turnours
0128 3/33 = 9% (507)* 6/42= 14% (450) 1/38 = 2.5 % (450) One with lung adenoma (450) 019 One with lung adenoma (406) 0115 One with sarcoma SC (406) 5/42= 12% (466) 0132 -
' Effective total=number of survivors in the group at the time of appearance of first turnour. - Number in parenthesis=average age of animals bearing turnours (in days). - a AAF = 0.5 ml of 2 % acetylaminofluorene SC on day I 5 of gestation. - ' 0.25 rmole of phorbol in phosphate-buffered saline IP, twice weekly, till weaning; then 0.5 @moleIP, twice weekly, for a total of 25 weeks; (cf Armuth, 1976, for similarly negative results with phorbol alone in C57BL/6 mice).
294
ARMUTH AND BERENBLUM
was definitely raised (8/74 = 11 %, as against 2/80 = 2.5% for males plus females: - significance : 0.02 < p
