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Lack of initiating and promoting activity of thiourea in rat liver foci bioassay
Cancer Letters,
410988)
245-
245
249
Elsevier Scientific Publishers Ireland Ltd.
LACK OF INITIATING AND PROMOTING ACTIVITY THIOUREA IN RAT LIVER FOCI BIOASSAY
OF
DORIS OESTERLE and ERHARD DEML Institute of Toxicology, Gesellschaft fiir Landstrasse 1, D-8042 Neuherberg IFR.G.I
Stfahlen-
und
Umweltforschung
mbH,
Ingolstddtet
(Received 5 November 1987) (Revised version received 13 June 1988) (Accepted 16 June 1988)
SUMMARY
Thiourea (TU) fails to enhance the incidence of foci deficient in adenosined’triphosphatase (ATPasel either by initiation or by promotion in a rat liver foci bioassay. To weanling female Sprague-Dawley rats, 3 x 200 or 3 x 500 mgl kg body wt of TU, respectively, were applied for initiation. One week later Clophen A 50, a technical mixture of polychlorinated biphenyls (PCBs) 2 x 10 mg/kg body wt were given twice weekly as promoting agent for 11 weeks. For promotion 0.2% of TU was administered with the drinking water for 51 days to rats of both sexes, and 0.1% and 0.05% of TU, respectively, for 70 days to females after initiation with 1 x 8 mg/kg body wt of diethylnitrosamine (DEN). Key words: Rat liver foci bioassay; Thiourea; Carcinogenicity.
INTRODUCTION
TU is widely used in the photographic and rubber industry, in flameretardant products, in the synthesis of pharmaceuticals and insecticides, and as a fungicide [l]. Long-term feeding of rats with TU shows that it is tumorigenic in various organs, preferentially in the thyroid and possibly in the liver [l- 31.In the thyroid the synthesis of thyroxin is inhibited by TU which entails hyperplasia with a tendency to malignant tumors [2,4]. The mechanism by which TU may induce adenomas in the liver is poorly understood. In most of the bacterial in vitro test systems no indication for genotoxicity was found [5, for literature see Ref. 61.A slight, but significant genotoxic activity of TU has been reported in mammalian cells [6]. Thus, at least weak initiating capacity is suggested. Besides a poor initiating potency, a promoting activity of TU may be responsible for hepatocarcinogenicity. Therefore we tested the initiating and promoting potency of TU in vivo by means of a rat liver foci bioassay. 0 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
0304-38351831t03.50
246 MATERIALSANDMETHODS
Sprague -Dawley rats of both sexes, 21-26 days old (inbreeding, Neuherberg, F.R.G.) were separated in 12 groups of 4-6 rats each. For initiation 2 groups of female rats received 3 x 200 and 3 x 500 mg TU/kg body wt (99.9% pure, SKW, Trostberg, F.R.G.1,respectively, in 2 ml water by gavage for 3 consecutive days. Six days later 10 mg Clophen A 5O/kg body wt (Bayer, Leverkusen, F.R.G.) in 2 ml olive oil was administered by gavage for promotion twice weekly for 11 consecutive weeks. Another 4 groups received either TU, Clophen A 50 or olive oil at doses given above. For evaluating the promoting activity of TU, groups of rats of both sexes each received 1 x 8 mg DEN/kg body wt DEN (analytical grade, 99% pure, Serva, Heidelberg, F.R.G.1 by gavage. One week later 0.2% TU in the drinking water was given for 51 days. Two groups of female rats received 0.1% and 0.05% TU in the drinking water for 70 days 1 week after initiation with 1 x 8 mg DEN/kg body wt. Another 2 groups of male and female rats were treated with DEN only. After 12 weeks the livers were examined for number and area of preneoplastic foci identified by the deficiency in ATPase in cryostat sections. One square centimeter per section and 8 cm2 per animal were screened by means of a semi-automatic picture analyzer (Videoplan, Kontron, Eching, F.R.G.1as described earlier [7]. RESULTS
The incidence of ATPase-deficient foci was not enhanced in the animals treated with 3 x 200 or 3 x 500 mg TU/kg body wt, respectively, given as an initiating agent as compared to controls treated with olive oil. In rats treated with TU and subsequently with PCBs as promoting agent foci incidence was up to about 1.6 per cm2,which was obtained also with PCBs only. In untreated rats about 0.2 and in TU-treated animals 0.1 foci/cm2 emerged (Table 1Al. Growth was normal and equaled that of controls (not shown), and rats treated with DEN only (Fig. 11. When TU was given as a promoting agent number and area of ATPasedeficient foci, induced by a single dose of DEN was strongly decreased. With 0.2% of TU in males the reduction in number amounted to 25%, in total area to 90% compared to DEN-treated controls (Table 1Bl. In females as compared to DEN-treated animals, number was reduced to 50% and total area to 30% with the concentrations of 0.2%. O.l”lb and 0.05% of TU (Table 1Bl. The body weight gain stagnated with 0.2% of TU and was 34% of the DEN-controls in males and 50% in females, at day 51, when TU was withdrawn (Fig. 11. With 0.1% and O.O5O/b of TU applied to females only over 70 days, body weight was lowered by 15% and IO%, respectively, compared to controls. The liver weight was reduced in these animals as well, whereas the liver to body weight ratio remained unchanged and amounted to about 3.2%.
247 TABLE 1 LACK OF INITIATING ACTIVITY RATS AND LACK OF PROMOTING SPRAGUE-DAWLEY RATS Treatment”
OF TU IN JUVENILE FEMALE ACTIVITY OF TU IN JUVENILE
ATPase-deficient
SPRAGUE-DAWLEY MALE AND FEMALE
islands
No./cm2
Area mm2/cm2
(A) Initiating schedule 200 mg TU + 10 mg PCBs 200 mg TU 500 mg TU + 10 mg PCBs 500 mg TU 10 mg PCBs Control (olive oil)
1.0 0.1 1.6 0.4 1.6 0.2
0.04 0.0004 0.03 0.002 0.02 0.0002
k 0.04 * 0.003 f 0.02 + 0.002 z!z0.01 f 0.0002
(B) Promoting scheduk? 8 mg DEN + 0.2% TU (0) 8 mg DEN + 0.11 TU (9) 8 mg DEN + 0.05% TU (0) 8 mg DEN (Q) 8 mg DEN + 0.2% TU (6) 8 mg DEN (6)
10.3 7.5 9.5 20.0 1.9 8.8
0.053 0.059 0.099 0.184 0.007 0.069
+ * f 2 2 r
+ f + f f +
+ f k f f +
0.8 0.1 0.8 0.2 0.9 0.1
1.8 1.3 3.9 4.9 1.1 3.8
0.012 0.011 0.050 0.048 0.004 0.029
“4 - 6 animals per group. bNo TU alone.
DISCUSSION
The tumorigenic activity of TU in rat liver as observed in a long term experiment [3] cannot be corroborated with our 12 weeks’ short term in vivo test. The marked reduction of foci incidence obtained in the promoting experiments could be not explained by the strong inhibitory effect of 0.20/bTU given over 51 days on body weight increase in both sexes. With 0.1% and 0.05% TU, given over 70 days to female rats, body weight was only about 15% and 10%. respectively, lower than in DEN-controls, although foci incidence was as low as in the group treated with 0.2% of TU. This indicates that growth and appearance of liver foci are not directly correlated. Tumors were observed in chronic experiments mainly in the thyroid [2,4], but also at various other sites, e.g. in the ear duct, the orbit [8], in the Zymbal and Meibomian gland of rats [9]. In one long term study adenomas in rat liver were reported [3]. Whereas malignant transformation of the thyroid may be related to an enhanced level of thyroid stimulating hormone (TSH) induced by TU, the mechanism of tumor formation in other organs is not understood. TU was not mutagenic in bacterial in vitro gene mutation assays [e.g. 5,101, and
248
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-c .$
400-
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!%
DEN
,-,
DEN
(1-0
DEN
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0.05
Z4,
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,i.’
.-.
0
DEN
+
0.1
‘7o TU
.-.
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DEN
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0.2
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.--.
?
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0.2
%
TU
n -.
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250-
200-
150-
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119
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Weeks Fig. 1. Development of body weight (g) in male and female Sprague-Dawley rats treated with 1 x 8 mg DEN/kg body wt for initiation, and with 0.2% TU for 51 days (males and females), or 0.1% and 0.05%, respectively, for 10 weeks (females) in the drinking water for promotion. For details see Materials and methods.
weakly mutagenic in Salmonella in the intraperitoneal host mediated assay using mice [lo]. In high doses TU evokes a weak effect on DNA repair in primary rat hepatocytes and on gene mutation in V79 cells [6]. In the alkaline elutionlrat hepatocyte assay DNA strand breaks have been observed [ll]. In a chronic study with TU in the dose range of 0.01 - l.OO/o,adenomas in rat liver emerged [3]. All animals at the dosage level of 0.25% and more died within 17 months. From 72 animals of the lower dose groups between 0.1%~and O.OlO/b, 29 survived the whole 2-year period, and only in these surviving rats were liver adenomas observed. Liver tumors did not emerge in any of the non-surviving rats with one exception, indicating that only a long term exposure to TU leads to tumor induction. Our results with a short term liver foci bioassay do not give any indication of an initiating or promoting activity of TU in rat liver.
249
ACKNOWLEDGEMENT
The expert technical assistance of Mrs. Carmen Spiller, Mrs. Marianne Berauer and cand. biol. Wolfgang Michl is gratefully acknowledged. The work was supported by Grant 160 040 017 from Umweltbundesamt, Berlin, F.R.G. REFERENCES IARC (1974) Some anti-thyroid and related substances, nitrofurans and industrial chemicals. IARC Monogr. Eval. Carcinog. Risk Chem. Man, 7,95- 109. Purves, H.D. and Griesbach, W.E. (19471 Studies on experimental goitre. VIII: Thyroid tumours in rats treated with thiourea. Br. J. Exp. Pathol., 28, 46-53. Fitzhugh, O.G. and Nelson, A.A. (19481 Liver tumors in rats fed thiourea or thioacetamide. Science, 108.626 - 628. Bielschowsky, F. (19491 The role of thyroxine deficiency in the formation of experimental tumors in the thyroid. Br. J. Cancer, 3.547-549. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (19751Detection of carcinogens as mutagens in the Salmon&r/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. USA, 72, 5135- 5139. 6 Ziegler-Skylakakis, K., Rossberger, S. and Andrae, U. (19851Thiourea induces DNA repair synthesis in primary rat hepatocyte cultures and gene mutations in V79 Chinese hamster cells. Arch. Toxicol., 58, 5-9. 7 Oesterle, D. and Deml, E. (19841Dose-dependent promoting effect of polychlorinated biphenyls on enzyme-altered islands in livers of adult and weanling rats. Carcinogenesis, 5,351- 355. 8 Rosin, A. and Ungar, H. (19571 Malignant tumors in the eyelids and in the auricular region of thiourea-treated rats. Cancer Res., 17,302-305. 9 Ungar, H. and Rosin, A. (19601The histogenesis of thiourea-induced carcinoma of the auditory duct sebaceous (Zymbal’sl glands in rats. Arch. De Vecchi Anat. Pathol., 31,419 - 430. 10 Simmon, V.F., Rosenkranz, H.S., Zeiger, E. and Poirier, L.A. (19791Mutagenic activity of chemical carcinogens and related compounds in the intraperitoneal host-mediated assay. J. Natl. Cancer Inst., 62, 911-918. 11 Sina, J.F., Bean, C.L., Dysart, G.R., Taylor, V.I. and Bradley, M.O. (19831 Evaluation of the alkaline elutionlrat hepatocyte assay as a predictor of carcinogenic/mutagenic potential. Mutat. Res., 113, 357-391.
410988)
245-
245
249
Elsevier Scientific Publishers Ireland Ltd.
LACK OF INITIATING AND PROMOTING ACTIVITY THIOUREA IN RAT LIVER FOCI BIOASSAY
OF
DORIS OESTERLE and ERHARD DEML Institute of Toxicology, Gesellschaft fiir Landstrasse 1, D-8042 Neuherberg IFR.G.I
Stfahlen-
und
Umweltforschung
mbH,
Ingolstddtet
(Received 5 November 1987) (Revised version received 13 June 1988) (Accepted 16 June 1988)
SUMMARY
Thiourea (TU) fails to enhance the incidence of foci deficient in adenosined’triphosphatase (ATPasel either by initiation or by promotion in a rat liver foci bioassay. To weanling female Sprague-Dawley rats, 3 x 200 or 3 x 500 mgl kg body wt of TU, respectively, were applied for initiation. One week later Clophen A 50, a technical mixture of polychlorinated biphenyls (PCBs) 2 x 10 mg/kg body wt were given twice weekly as promoting agent for 11 weeks. For promotion 0.2% of TU was administered with the drinking water for 51 days to rats of both sexes, and 0.1% and 0.05% of TU, respectively, for 70 days to females after initiation with 1 x 8 mg/kg body wt of diethylnitrosamine (DEN). Key words: Rat liver foci bioassay; Thiourea; Carcinogenicity.
INTRODUCTION
TU is widely used in the photographic and rubber industry, in flameretardant products, in the synthesis of pharmaceuticals and insecticides, and as a fungicide [l]. Long-term feeding of rats with TU shows that it is tumorigenic in various organs, preferentially in the thyroid and possibly in the liver [l- 31.In the thyroid the synthesis of thyroxin is inhibited by TU which entails hyperplasia with a tendency to malignant tumors [2,4]. The mechanism by which TU may induce adenomas in the liver is poorly understood. In most of the bacterial in vitro test systems no indication for genotoxicity was found [5, for literature see Ref. 61.A slight, but significant genotoxic activity of TU has been reported in mammalian cells [6]. Thus, at least weak initiating capacity is suggested. Besides a poor initiating potency, a promoting activity of TU may be responsible for hepatocarcinogenicity. Therefore we tested the initiating and promoting potency of TU in vivo by means of a rat liver foci bioassay. 0 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
0304-38351831t03.50
246 MATERIALSANDMETHODS
Sprague -Dawley rats of both sexes, 21-26 days old (inbreeding, Neuherberg, F.R.G.) were separated in 12 groups of 4-6 rats each. For initiation 2 groups of female rats received 3 x 200 and 3 x 500 mg TU/kg body wt (99.9% pure, SKW, Trostberg, F.R.G.1,respectively, in 2 ml water by gavage for 3 consecutive days. Six days later 10 mg Clophen A 5O/kg body wt (Bayer, Leverkusen, F.R.G.) in 2 ml olive oil was administered by gavage for promotion twice weekly for 11 consecutive weeks. Another 4 groups received either TU, Clophen A 50 or olive oil at doses given above. For evaluating the promoting activity of TU, groups of rats of both sexes each received 1 x 8 mg DEN/kg body wt DEN (analytical grade, 99% pure, Serva, Heidelberg, F.R.G.1 by gavage. One week later 0.2% TU in the drinking water was given for 51 days. Two groups of female rats received 0.1% and 0.05% TU in the drinking water for 70 days 1 week after initiation with 1 x 8 mg DEN/kg body wt. Another 2 groups of male and female rats were treated with DEN only. After 12 weeks the livers were examined for number and area of preneoplastic foci identified by the deficiency in ATPase in cryostat sections. One square centimeter per section and 8 cm2 per animal were screened by means of a semi-automatic picture analyzer (Videoplan, Kontron, Eching, F.R.G.1as described earlier [7]. RESULTS
The incidence of ATPase-deficient foci was not enhanced in the animals treated with 3 x 200 or 3 x 500 mg TU/kg body wt, respectively, given as an initiating agent as compared to controls treated with olive oil. In rats treated with TU and subsequently with PCBs as promoting agent foci incidence was up to about 1.6 per cm2,which was obtained also with PCBs only. In untreated rats about 0.2 and in TU-treated animals 0.1 foci/cm2 emerged (Table 1Al. Growth was normal and equaled that of controls (not shown), and rats treated with DEN only (Fig. 11. When TU was given as a promoting agent number and area of ATPasedeficient foci, induced by a single dose of DEN was strongly decreased. With 0.2% of TU in males the reduction in number amounted to 25%, in total area to 90% compared to DEN-treated controls (Table 1Bl. In females as compared to DEN-treated animals, number was reduced to 50% and total area to 30% with the concentrations of 0.2%. O.l”lb and 0.05% of TU (Table 1Bl. The body weight gain stagnated with 0.2% of TU and was 34% of the DEN-controls in males and 50% in females, at day 51, when TU was withdrawn (Fig. 11. With 0.1% and O.O5O/b of TU applied to females only over 70 days, body weight was lowered by 15% and IO%, respectively, compared to controls. The liver weight was reduced in these animals as well, whereas the liver to body weight ratio remained unchanged and amounted to about 3.2%.
247 TABLE 1 LACK OF INITIATING ACTIVITY RATS AND LACK OF PROMOTING SPRAGUE-DAWLEY RATS Treatment”
OF TU IN JUVENILE FEMALE ACTIVITY OF TU IN JUVENILE
ATPase-deficient
SPRAGUE-DAWLEY MALE AND FEMALE
islands
No./cm2
Area mm2/cm2
(A) Initiating schedule 200 mg TU + 10 mg PCBs 200 mg TU 500 mg TU + 10 mg PCBs 500 mg TU 10 mg PCBs Control (olive oil)
1.0 0.1 1.6 0.4 1.6 0.2
0.04 0.0004 0.03 0.002 0.02 0.0002
k 0.04 * 0.003 f 0.02 + 0.002 z!z0.01 f 0.0002
(B) Promoting scheduk? 8 mg DEN + 0.2% TU (0) 8 mg DEN + 0.11 TU (9) 8 mg DEN + 0.05% TU (0) 8 mg DEN (Q) 8 mg DEN + 0.2% TU (6) 8 mg DEN (6)
10.3 7.5 9.5 20.0 1.9 8.8
0.053 0.059 0.099 0.184 0.007 0.069
+ * f 2 2 r
+ f + f f +
+ f k f f +
0.8 0.1 0.8 0.2 0.9 0.1
1.8 1.3 3.9 4.9 1.1 3.8
0.012 0.011 0.050 0.048 0.004 0.029
“4 - 6 animals per group. bNo TU alone.
DISCUSSION
The tumorigenic activity of TU in rat liver as observed in a long term experiment [3] cannot be corroborated with our 12 weeks’ short term in vivo test. The marked reduction of foci incidence obtained in the promoting experiments could be not explained by the strong inhibitory effect of 0.20/bTU given over 51 days on body weight increase in both sexes. With 0.1% and 0.05% TU, given over 70 days to female rats, body weight was only about 15% and 10%. respectively, lower than in DEN-controls, although foci incidence was as low as in the group treated with 0.2% of TU. This indicates that growth and appearance of liver foci are not directly correlated. Tumors were observed in chronic experiments mainly in the thyroid [2,4], but also at various other sites, e.g. in the ear duct, the orbit [8], in the Zymbal and Meibomian gland of rats [9]. In one long term study adenomas in rat liver were reported [3]. Whereas malignant transformation of the thyroid may be related to an enhanced level of thyroid stimulating hormone (TSH) induced by TU, the mechanism of tumor formation in other organs is not understood. TU was not mutagenic in bacterial in vitro gene mutation assays [e.g. 5,101, and
248
G
450-
-c .$
400-
d
!%
DEN
,-,
DEN
(1-0
DEN
z mo
3m-
300-
t
0.05
Z4,
TU
,i.’
.-.
0
DEN
+
0.1
‘7o TU
.-.
d
DEN
t
0.2
%
TU
.--.
?
DEN
+
0.2
%
TU
n -.
’
0/
250-
200-
150-
loo-
m-
o-
119
4,
5I
6,
7I
8I
9I
10I
11I
121
Weeks Fig. 1. Development of body weight (g) in male and female Sprague-Dawley rats treated with 1 x 8 mg DEN/kg body wt for initiation, and with 0.2% TU for 51 days (males and females), or 0.1% and 0.05%, respectively, for 10 weeks (females) in the drinking water for promotion. For details see Materials and methods.
weakly mutagenic in Salmonella in the intraperitoneal host mediated assay using mice [lo]. In high doses TU evokes a weak effect on DNA repair in primary rat hepatocytes and on gene mutation in V79 cells [6]. In the alkaline elutionlrat hepatocyte assay DNA strand breaks have been observed [ll]. In a chronic study with TU in the dose range of 0.01 - l.OO/o,adenomas in rat liver emerged [3]. All animals at the dosage level of 0.25% and more died within 17 months. From 72 animals of the lower dose groups between 0.1%~and O.OlO/b, 29 survived the whole 2-year period, and only in these surviving rats were liver adenomas observed. Liver tumors did not emerge in any of the non-surviving rats with one exception, indicating that only a long term exposure to TU leads to tumor induction. Our results with a short term liver foci bioassay do not give any indication of an initiating or promoting activity of TU in rat liver.
249
ACKNOWLEDGEMENT
The expert technical assistance of Mrs. Carmen Spiller, Mrs. Marianne Berauer and cand. biol. Wolfgang Michl is gratefully acknowledged. The work was supported by Grant 160 040 017 from Umweltbundesamt, Berlin, F.R.G. REFERENCES IARC (1974) Some anti-thyroid and related substances, nitrofurans and industrial chemicals. IARC Monogr. Eval. Carcinog. Risk Chem. Man, 7,95- 109. Purves, H.D. and Griesbach, W.E. (19471 Studies on experimental goitre. VIII: Thyroid tumours in rats treated with thiourea. Br. J. Exp. Pathol., 28, 46-53. Fitzhugh, O.G. and Nelson, A.A. (19481 Liver tumors in rats fed thiourea or thioacetamide. Science, 108.626 - 628. Bielschowsky, F. (19491 The role of thyroxine deficiency in the formation of experimental tumors in the thyroid. Br. J. Cancer, 3.547-549. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (19751Detection of carcinogens as mutagens in the Salmon&r/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. USA, 72, 5135- 5139. 6 Ziegler-Skylakakis, K., Rossberger, S. and Andrae, U. (19851Thiourea induces DNA repair synthesis in primary rat hepatocyte cultures and gene mutations in V79 Chinese hamster cells. Arch. Toxicol., 58, 5-9. 7 Oesterle, D. and Deml, E. (19841Dose-dependent promoting effect of polychlorinated biphenyls on enzyme-altered islands in livers of adult and weanling rats. Carcinogenesis, 5,351- 355. 8 Rosin, A. and Ungar, H. (19571 Malignant tumors in the eyelids and in the auricular region of thiourea-treated rats. Cancer Res., 17,302-305. 9 Ungar, H. and Rosin, A. (19601The histogenesis of thiourea-induced carcinoma of the auditory duct sebaceous (Zymbal’sl glands in rats. Arch. De Vecchi Anat. Pathol., 31,419 - 430. 10 Simmon, V.F., Rosenkranz, H.S., Zeiger, E. and Poirier, L.A. (19791Mutagenic activity of chemical carcinogens and related compounds in the intraperitoneal host-mediated assay. J. Natl. Cancer Inst., 62, 911-918. 11 Sina, J.F., Bean, C.L., Dysart, G.R., Taylor, V.I. and Bradley, M.O. (19831 Evaluation of the alkaline elutionlrat hepatocyte assay as a predictor of carcinogenic/mutagenic potential. Mutat. Res., 113, 357-391.