- Email: [email protected]
Enhancement by non-mutagenic pesticides of GST-P positive hepatic foci development initiated with diethylnitrosamine in the rat
Cancer Letters, 72 (1993) !i9-64 Elsevier Scientific Publishers Ireland
59 Ltd.
Enhancement by non-mutagenic pesticides of GST-P positive hepatic foci dlevelopment initiated with diethylnitrosamine in the rat Tom Hoshiya, Ryohei Hasegawa, Kazuo Hakoi, Lin Cui, Tadashi Ogiso, Ricardo Cabral and Nobuyuki Ito First Department of Pathobgy. Nagoya City University Medical School. Nagoya (Japan) (Received 19 February 1992) (Revision received 22 April 1993) (Accepted 14 May 1993)
Summary
Keywords: pesticides; hepatocarcinogenesis; P positive foci; rapid bioassay; F344 rat
The potential hepatocarcinogenicity of seven pesticides was examined using a rapid bioassay based on the induction of glutathione S-transferase placental form positive foci in the rat liver. Rats were initially injected with diethylnitrosamine and two weeks later were fed on diet supplemented with one of the pesticides for 6 weeks and then killed; all rats were subjected to a partial hepatectomy at week 3. Positive results were seen with chlorobenzilate (2000 ppm), vinclozolin (2000 ppm), malathion (4000 ppm), tecnazene (2000 ppm) and isoproturon (2000 ppm). S.S,Stributylphosphorotrithioate (DEF, 200 ppm) and dicloran (2000 ppm) were negative in both number and area analyses. Although chlorobenzilate is carcinogenic in mice, malathion and vinclozolin have been reported as non-carcinogens in both rats and mice. Since th.e present system is based on the two-stage carcinogenesis hypothesis, it is possible that the chemicals showing positive results in this system possess at least tumor-promoting activity in the rat liver. This is very significant, as most carcinogens show tumor-promoting activity in their target organs. Correspondence to: Nobuyuki Ito, First Department Pathology, Nagoya City University Medical School, Kawasumi, Mizuho-cho, Misuho-ku, Nagoya 467. Japan. 0304-3835/93/$06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
of 1
Ire
GST-
Introduction Investigation of possible environmentally derived influences in animal experiments has established that a vast array of compounds are capable of playing a role in tumorigenesis; and detection and appropriate regulation of these compounds are of prime importance for the prevention of neoplasia in humans. Agricultural chemicals are recognized as possible environmental toxic or carcinogenic agents [1,2]. Not only workers in industry and agriculture but also the general population may be exposed to those chemicals from foods. Evaluation of potential carcinogenic or tumor-modifying activities of agricultural chemicals is therefore clearly very important. In our laboratory a medium-term bioassay system of only 8 weeks’ duration utilizing rat liver glutathione S-transferase placental form (GST-P) positive foci as end-point markers has been introduced to bridge a long-term carcinogenicity bioassay and in vitro screening tests; the reliability of the system is reviewed elsewhere 13-61. Using this system, we have examined possible carcinogenicity of a variety of pesticides. The criteria used to select the compounds were (i) evidence of toxicity as described in the literature, (ii) widespread use, (iii) Ltd.
60
chemical potential, suspected
structure suggestive of carcinogenic or (iv) structural similarity to other agents [7].
Materials and Methods
Chemicals Diethylnitrosamine (DEN) was obtained from Tokyo Chemical Company, Tokyo. The pesticides used were either obtained from commercial sources or received as gifts from the following: vinclozolin from BASF India Ltd., Bombay; malathion from Cheminova, Lemvig, Denmark; S&S-tributylphosphorotrithioate (DEF) a gift from Dr. Cabral; chlorobenzilate from Nippon Kayaku Co. Ltd., Tokyo; tecnazene from Aldrich Chemical Company, Milwaukee, Wisconsin; isoproturon from Hoechst AG, Frankfurt; and dicloran from Wako Pure Chemical Industry, Ltd.. Osaka.
Animals and treatments A total of 194 male F344 rats (Charles River Japan Inc., Atsugi), 6 weeks old at commencement, were maintained on basal diet (Oriental M,
0
2
Oriental Yeast Company, Tokyo) ad libitum and housed in plastic cages in an air-conditioned room at 24 f 2°C and 55 f 5% humidity with a 12 h/l2 h light-dark cycle. The animals were divided into three groups, as shown in Fig. 1. Group 1 was given a single i.p. injection of DEN (200 mgikg body weight) dissolved in saline to initiate hepatocarcinogenesis and, after a 2 week recovery period on basal diet, received one of the test compounds for the next weeks. Animals were subjected to two-thirds partial hepatectomy (PH) at week 3 to maximize any interaction between cell proliferation and the effects of the compounds tested. Group 2 was given DEN and PH in the same manner as group 1 without any test compound. Group 3 rats were injected with saline instead of DEN solution and then subjected to test compounds and PH. Test compounds were given in the basal diet at the following concentrations: vinclozolin, 2000 ppm; malathion, 4000 ppm; DEF, 200 ppm; chlorobenzilate, 2000 ppm; tecnazene, 2000 ppm; isoproturon. 2000 ppm; and dicloran, 2000 ppm. The doses of test compounds were chosen on the basis of preliminary long-term tests or toxicity data, if available [8-141.
8 weeks
3
Group 1
3
: DEN, 200 mg/kg i.p.
W
: Test chemical
&
: Saline, i.p.
I
: No treatment
V
: Two-thirds partial hepatectomy
t
Fig. 1.
Experimental
protocol
S
: Sacrifice rats for GST-P immunohistochemistry
61
All surviving animals were sacrificed at week 8, livers were immediate1.y excised, and sections 2-3 mm thick were cut with a razor blade. Three slices, one each from the right posterior, caudate and right anterior lobes, were fixed in ice-cold acetone for immunohistochemical examination of GST-P. Tissue processing and GST-P positive foci analysis
GST-P immunohistochemistry was performed, as previously described [6,15]. The numbers and the areas of GST-P positive foci of more than 0.2 mm in diameter and th.e total areas of the liver sections examined were measured using a video image processor (VIP-2 1C, Olympus Company, Tokyo), as previously reported [16]. The results were assessed by comparing the values of foci between group 1 (DEN test compounds) and group 2 (DEN alone). Group 3 animals served to assay the potential of the test chemicals to induce GST-P positive foci without prior DE.N. Statistical analysis
Statistical analysis was carried out using Student’s t-test and Welch’s t-test in combination with the F-test for means [17]. A positive result was
lkble
I.
Group
Final body and liver weights
Results
The numbers of survivors in the experimental groups is shown in Table I. Overall mortality following PH was 2.6%, and no treatment-related mortality was seen. Final body and liver weights and data for food consumption during the period of feeding of the test chemical are summarized in Table I. Body weights as compared with the DENonly group were significantly decreased in the vinclozolin and DEF groups. Absolute liver weights were increased in the malathion, chlorobenzilate, tecnazene, isoproturon and diclorantreated groups, and relative liver weight (to body weight) was increased in all groups. In the noninitiated groups, body weight was slightly higher than those in the initiated groups, and the relative liver weight was also increased similarly to the initiated groups. Numbers and areas of GST-P positive foci per unit area of liver section after treatment with and
of rats.
No. of rats
Treatment
considered to be obtained when there was an increase at P < 0.05 in either number or area of foci between groups 1 and 2.
Body weight (g)
Liver weight Absolute
(g)
Liver/body
(%)
1. DEN I6 16 16 16 14 15 I4
254 260 242 266 273 270 271
2. DEN alone
15
267 f
19
3. Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon Dicloran
10 9 10 8 10 10 10
268 286 254 284 282 288 274
I6 IO 9 20 15 12 I5
Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon Dicloran
Significantly
different
from group
2 at
f l5C f I7 f 15a f 9 f 10 ZIZ13 f 14
f f f f f f f
8.8 9.9 8.1 10.9 II.5 10.6 12.5
8.4 f 9.0 10.8 8.3 12.7 12.1 10.9 12.1
3.49 3.80 3.35 4.09 4.19 3.90 4.62
f f * f f + f
0.09a 0.45a 0.22b 0.15” 0.14” 0.18” 0.28a
1.0
3.13
l
0.16
0.8 0.6 0.6 1.1 0.5 0.5 0.6
3.36 3.77 3.28 4.47 4.30 3.79 4.42
f 0.15 l 0.14 + 0.22 f 0.22 f 0.21 f 0.22 f 0.16
f 0.6 f 1.2” f 0.7 f 0.6” l 0.7a f 0.8= f 0.9a
f f f f f f f
aP < 0.001, bP < 0.01or 'P < 0.05, respectively.
62
Table II.
Results
Group
of GST-P
Treatment
positive
foci analysis. No. of rats
GST-P positive
foci
Number
(n/cm’)
Area (mm’icm’)
16.08 12.81 6.92 13.89 12.70 18.76 6.60
4.62’ 4.07h 2.43 3.29” 2.46’ 2.94” 2.59
1.48 I.11 0.48 1.27 1.04 1.63 0.50
1. DEN Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon Dicloran 2. DEN alone 3. Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon Dicloran
16 16 16 16 14 15 14
f f f f f f f
f f + f f f f
0.49” 0.49c 0.20’ 0.39” 0.27b 0.37” 0.33
15
8.32 f 3.71
0.72 f 0.31
IO 9 IO 8 IO IO IO
0 0 0 0 0 0.22 f 0.27 0
0 0 0 0 0 0.01 f 0.02 0
GST-P positive foci larger than 0.2 mm in diameter were counted. Significantly different from group 2 at “P < 0.001, bP < 0.01 or ‘P < 0.05, respectively
without DEN initiation are summarized in Table II. The number of foci in the control group was 8.32/cm2 and the area was 0.72 mm2icm’ Vinclozolin, malathion, chlorobenzilate. tecnazene and isoproturon increased GST-P positive foci development over the control levels in both parameters. Dicloran did not affect foci development, and DEF rather decreased the area of foci. In the non-DEN-initiated groups, only isoproturon induced a small number of GST-P positive foci larger than 0.2 mm in diameter. Discussion Since this assay model is based on the two-step hypothesis of carcinogenesis and on the induction of pre-neoplastic hepatocyte lesions, it primarily provides information on whether a test compound possesses hepato-promoting activity in the rat [6]. However, we think the carcinogenic potential of chemicals can be best assessed by detection of their promoting activity based on the following concepts: (i) most carcinogens possess both tumorinitiating and tumor-promoting activities, and (ii)
carcinogenicity and organotropism of carcinogenic agents might be determined mainly by their tumor-promoting activity. Actually more than 90% of the liver carcinogens could be positively detected by this system, although assessment of carcinogens other than established hepatocarcinogens gave less positive results [3-61. In the present study, five of the seven pesticides were positive: chlorobenzilate, vinclozolin, malathion, tecnazene, and isoproturon. Dicloran was negative in terms of both the number and area of GST-P positive foci, and DEF decreased the area of foci under the present experimental conditions. Dicloran increased the liver weight and was negative in this system. DEF exerted the strongest general toxicity among the seven chemicals; it rather decreased the foci induction at 200 ppm, the lowest dose level among the seven chemicals. Thus the positivities for the pesticides examined were not directly correlated to general and/or liver toxicity of the chemicals as evaluated by the weight data, although some reports indicate that chemicals that increased liver weight enhanced the development of pre-neoplastic foci of the liver [ 181.
63
Table III.
Comparison
01‘ positivity.
Chemical
Chlor.. inated
Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon
+ -
Dicloran
+ + +
Mutagenicity
? _ ? ? -
Carcinogenicity
Liver system
-
+ +
? + ? ? ?
+ + + -
+ Positive or yes; - negative or no; ? unknown. Mutagenicity in the Ames test. References: [9-161
The results are co:mpared with their reported mutagenicity in the Ames test and reported carcinogenicity (Table III). Mutagenicity in the Ames test is negative for vinclozolin, malathion, chlorobenzilate, and dicloran, and unknown to us for the other three compounds [8-14,19,20]. Among the seven pesticides, chlorobenzilate has been reported to be carcinogenic, but only in the mouse liver [8-lo]. Similar cases of non-mutagenic carcinogens are more commonly observed for the other chlorinated compounds [ 19,201. The positive results for malathion and vinclozolin are interesting, as these two chemicals have been evaluated as ‘non-carcinogens’ in both rats and mice and non-mutagenic in the Ames test [13,19.20]. Carcinogenicity for DEF, dicloran, tecnazene and isoproturon has not 1.~11~evaluated as far as we know. Malathion has been reported as ‘noncarcinogenic’ in NTP technical reports [ 11,121. However, it is worth noting that this agent has demonstrated significant enhancement of the development of several types of tumor in both rats and mice, and the combined incidence of hepatocellular carcinomas and neoplastic nodules in male mice showed a significant linear trend when compared with either the matched controls (P = 0.041) or the pooled controls (P = 0.019) when administered at concentrations of 8000 and 16 000 ppm in the diet. Although liver carcinogenicity was not evident for malathion in either Osborne Mendel or Fischer rats, our results suggest that malathion possesses tumor-promoting activity in the liver. Vinclozolin has also been reported to be
non-carcinogenic by the FAO and WHO [13]; however, an apparent increase in liver weight was noted for both rats and mice to which the chemical was administered at concentrations of up to 4370 ppm in the diet. The level of positivity in this system was almost the same as that of phenobarbital at 0.05% in the diet. Phenobarbital has been considered a typical hepatopromoter and recently evaluated as a liver carcinogen [21]. There seems to be a discrepancy, therefore, between the reported hepatocarcinogenicity in the rat and the positivity in the present system for three chemicals. We consider, however, that these three compounds are possible hepatopromoters in the rat, and this activity has not been fully demonstrated in the long-term carcinogenicity study in this species. It is possible to conclude that chemicals exerting a positive result in this system might possess hepatopromoting activity even if the hepatocarcinogenicity has not been demonstrated in a longterm study in the rat. In the strategy for cancer prevention, we believe that a battery of rapid tests for carcinogenicity in the liver has an important role to play. Acknowledgments
The authors would like to express their gratitude to the late Dr. Kiyomi Sato, Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki, for generously providing GST-P antibody. This work was supported in part by grants in aid for cancer research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan, and grants from the Society for the Promotion of Pathology of Nagoya and the Experimental Pathological Research Association of Nagoya, Japan. Ricardo Cabral was supported by the Program for the Invitation of Foreign Scientists to Japanese Institutes by the Foundation for Promotion of Cancer Research, Tokyo, Japan. References 1
Ferber. A. and Cabral, R. (1991) Toxic epidemics caused by alimentary exposure to pesticides: a review. Food Additives Contam.. 8. 755-776.
64
6
8
International Agency for Research on Cancer (1991) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol. 53: Occupational Exposures in Insecticide Application, and Some. Pesticides. IARC Scientific Publications, Lyon. Ito, N., Tsuda, H., Tatematsu, M., Inoue, T., Tagawa. Y.. Aoki, T., Uwagawa, S., Kagawa, M., Ogiso. T.. Masui, T., Imaida, K., Fukushima, S. and Asamoto, M. (1988) Enhancing effect of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in rats: an approach for a new mediumterm bioassay system. Carcinogenesis, 9, 387-394. Hasegawa, R. and Ito, N. (1992) Liver medium-term bioassay in rats for screening of carcinogens and modifying factors in hepatocarcinogenesis. Food Chem. Toxicol., 30, 979-992. Ito, N., Shirai, T. and Hasegawa, R. (1992) Medium-term bioassay for carcinogens. In: Mechanisms of Carcinogenesis in Risk Assessment, pp. 353-388. Editors: H. D.B. McGregor and A.J. Vainio, P.N. Magee. McMichael. IARC, Lyon. Ito, N., Imaida, K., Hasegawa, R. and Tsuda, H. (1989) Rapid bioassay methods for carcinogens and modifiers of hepatocarcinogenesis. CRC Crit. Rev. Toxicol.. 19, 385-415. Cabral, R., Hoshiya, T., Hakoi, K., Hasegawa, R., Fukushima, S. and Ito, N. (1991) A rapid in vivo bioassay for the carcinogenicity of pesticides, Tumori, 77, 185-188. National Toxicology Program (1978) Toxicology and carcinogenesis studies of chlorobenzilate in rats (Technical Report Series no. 75). U.S. Department of Health and Human Services, Public Health Service, Research Triangle Park. International Agency for Research on Cancer (1983) IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans: Miscellaneous Pesticides. IARC, Lyon. International Agency for Research on Cancer (1987) IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man: Some Organochlorine Pesticides, pp. 75-81. IARC, Lyon. National Toxicology Program (1978) Toxicology and carcinogenesis studies of malathion in rats (Technical Report
12
16
I7
18
19
20
21
Series no. 24). U.S. Department of Health and Human Services, Public Health Service. Research Triangle Park. National Toxicology Program (1979) Toxicology and carcinogenesis studies of malathion in F344M rats and B6C3Ft mice (Technical Report Series no. 192). U.S. Department of Health and Human Services, Public Health Service, Research Triangle Park. FAO-WHO (1986) Pesticide Residues in Food: 1986 Evaluations. pp. 193-207. World Health Organization, Geneva. FAO-WHO (1977) Pesticide Residues in Food: Report. 1977, pp. 24-26. World Health Organization, Geneva. Tatematsu, M., Mera, Y., Ito. N., Satoh. K. and Sato. K. (1985) Relative merits of immunohistochemical demonstration of placental A, B and C forms of glutathione Stransferase and histochemical demonstration of yglutamyl transferase as markers of altered foci during liver carcinogenesis in rats. Carcinogenesis. 6, 162 1- 1626. Tatematsu, M., Takano, T., Hasegawa, R.. Imaida, K., Nakanowatari, J. and Ito, N. (1980) A sequential quantitative study of the reversibility or irreversibility of liver hyperplastic nodules in rats exposed to hepatocarcinogens. Gann, 71, 843-1755. Gad, S.C and Weil, C.S. (1982) Statistics for Toxicologists. In: Principles and Methods of Toxicology. pp. 273-320. Editor: A.W. Hayes. Raven Press, New York. Tatematsu, M.. Nakanishi, K., Murasaki, C., Miyata. Y., Hirose, M. and Ito. N. (1979) Enhancing effect of inducers of liver microsomal enzymes on induction of hyperplastic liver nodules by N-2-fluorenylacetamide in rats. J. Nat. Cancer Inst.. 63, 1411-1416. Zeiger, E. (1987) Carcinogenicity of mutagens: predictive capability of the Sulmone/lu mutagenesis assay for rodent carcinogenicity. Cancer Res., 47. I287- 1296. Ashby, J. and Tennant, R.W. (1991) Definitive relationship among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP. Mutat. Res.. 257. 229-306. International Agency for Research on Cancer (1987) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Overall Evaluations of Carcinogenicity: an Updating of IARC Monographs Vols. 1 to 42, Suppl. 7. IARC Scientific Publications, Lyon.
59 Ltd.
Enhancement by non-mutagenic pesticides of GST-P positive hepatic foci dlevelopment initiated with diethylnitrosamine in the rat Tom Hoshiya, Ryohei Hasegawa, Kazuo Hakoi, Lin Cui, Tadashi Ogiso, Ricardo Cabral and Nobuyuki Ito First Department of Pathobgy. Nagoya City University Medical School. Nagoya (Japan) (Received 19 February 1992) (Revision received 22 April 1993) (Accepted 14 May 1993)
Summary
Keywords: pesticides; hepatocarcinogenesis; P positive foci; rapid bioassay; F344 rat
The potential hepatocarcinogenicity of seven pesticides was examined using a rapid bioassay based on the induction of glutathione S-transferase placental form positive foci in the rat liver. Rats were initially injected with diethylnitrosamine and two weeks later were fed on diet supplemented with one of the pesticides for 6 weeks and then killed; all rats were subjected to a partial hepatectomy at week 3. Positive results were seen with chlorobenzilate (2000 ppm), vinclozolin (2000 ppm), malathion (4000 ppm), tecnazene (2000 ppm) and isoproturon (2000 ppm). S.S,Stributylphosphorotrithioate (DEF, 200 ppm) and dicloran (2000 ppm) were negative in both number and area analyses. Although chlorobenzilate is carcinogenic in mice, malathion and vinclozolin have been reported as non-carcinogens in both rats and mice. Since th.e present system is based on the two-stage carcinogenesis hypothesis, it is possible that the chemicals showing positive results in this system possess at least tumor-promoting activity in the rat liver. This is very significant, as most carcinogens show tumor-promoting activity in their target organs. Correspondence to: Nobuyuki Ito, First Department Pathology, Nagoya City University Medical School, Kawasumi, Mizuho-cho, Misuho-ku, Nagoya 467. Japan. 0304-3835/93/$06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
of 1
Ire
GST-
Introduction Investigation of possible environmentally derived influences in animal experiments has established that a vast array of compounds are capable of playing a role in tumorigenesis; and detection and appropriate regulation of these compounds are of prime importance for the prevention of neoplasia in humans. Agricultural chemicals are recognized as possible environmental toxic or carcinogenic agents [1,2]. Not only workers in industry and agriculture but also the general population may be exposed to those chemicals from foods. Evaluation of potential carcinogenic or tumor-modifying activities of agricultural chemicals is therefore clearly very important. In our laboratory a medium-term bioassay system of only 8 weeks’ duration utilizing rat liver glutathione S-transferase placental form (GST-P) positive foci as end-point markers has been introduced to bridge a long-term carcinogenicity bioassay and in vitro screening tests; the reliability of the system is reviewed elsewhere 13-61. Using this system, we have examined possible carcinogenicity of a variety of pesticides. The criteria used to select the compounds were (i) evidence of toxicity as described in the literature, (ii) widespread use, (iii) Ltd.
60
chemical potential, suspected
structure suggestive of carcinogenic or (iv) structural similarity to other agents [7].
Materials and Methods
Chemicals Diethylnitrosamine (DEN) was obtained from Tokyo Chemical Company, Tokyo. The pesticides used were either obtained from commercial sources or received as gifts from the following: vinclozolin from BASF India Ltd., Bombay; malathion from Cheminova, Lemvig, Denmark; S&S-tributylphosphorotrithioate (DEF) a gift from Dr. Cabral; chlorobenzilate from Nippon Kayaku Co. Ltd., Tokyo; tecnazene from Aldrich Chemical Company, Milwaukee, Wisconsin; isoproturon from Hoechst AG, Frankfurt; and dicloran from Wako Pure Chemical Industry, Ltd.. Osaka.
Animals and treatments A total of 194 male F344 rats (Charles River Japan Inc., Atsugi), 6 weeks old at commencement, were maintained on basal diet (Oriental M,
0
2
Oriental Yeast Company, Tokyo) ad libitum and housed in plastic cages in an air-conditioned room at 24 f 2°C and 55 f 5% humidity with a 12 h/l2 h light-dark cycle. The animals were divided into three groups, as shown in Fig. 1. Group 1 was given a single i.p. injection of DEN (200 mgikg body weight) dissolved in saline to initiate hepatocarcinogenesis and, after a 2 week recovery period on basal diet, received one of the test compounds for the next weeks. Animals were subjected to two-thirds partial hepatectomy (PH) at week 3 to maximize any interaction between cell proliferation and the effects of the compounds tested. Group 2 was given DEN and PH in the same manner as group 1 without any test compound. Group 3 rats were injected with saline instead of DEN solution and then subjected to test compounds and PH. Test compounds were given in the basal diet at the following concentrations: vinclozolin, 2000 ppm; malathion, 4000 ppm; DEF, 200 ppm; chlorobenzilate, 2000 ppm; tecnazene, 2000 ppm; isoproturon. 2000 ppm; and dicloran, 2000 ppm. The doses of test compounds were chosen on the basis of preliminary long-term tests or toxicity data, if available [8-141.
8 weeks
3
Group 1
3
: DEN, 200 mg/kg i.p.
W
: Test chemical
&
: Saline, i.p.
I
: No treatment
V
: Two-thirds partial hepatectomy
t
Fig. 1.
Experimental
protocol
S
: Sacrifice rats for GST-P immunohistochemistry
61
All surviving animals were sacrificed at week 8, livers were immediate1.y excised, and sections 2-3 mm thick were cut with a razor blade. Three slices, one each from the right posterior, caudate and right anterior lobes, were fixed in ice-cold acetone for immunohistochemical examination of GST-P. Tissue processing and GST-P positive foci analysis
GST-P immunohistochemistry was performed, as previously described [6,15]. The numbers and the areas of GST-P positive foci of more than 0.2 mm in diameter and th.e total areas of the liver sections examined were measured using a video image processor (VIP-2 1C, Olympus Company, Tokyo), as previously reported [16]. The results were assessed by comparing the values of foci between group 1 (DEN test compounds) and group 2 (DEN alone). Group 3 animals served to assay the potential of the test chemicals to induce GST-P positive foci without prior DE.N. Statistical analysis
Statistical analysis was carried out using Student’s t-test and Welch’s t-test in combination with the F-test for means [17]. A positive result was
lkble
I.
Group
Final body and liver weights
Results
The numbers of survivors in the experimental groups is shown in Table I. Overall mortality following PH was 2.6%, and no treatment-related mortality was seen. Final body and liver weights and data for food consumption during the period of feeding of the test chemical are summarized in Table I. Body weights as compared with the DENonly group were significantly decreased in the vinclozolin and DEF groups. Absolute liver weights were increased in the malathion, chlorobenzilate, tecnazene, isoproturon and diclorantreated groups, and relative liver weight (to body weight) was increased in all groups. In the noninitiated groups, body weight was slightly higher than those in the initiated groups, and the relative liver weight was also increased similarly to the initiated groups. Numbers and areas of GST-P positive foci per unit area of liver section after treatment with and
of rats.
No. of rats
Treatment
considered to be obtained when there was an increase at P < 0.05 in either number or area of foci between groups 1 and 2.
Body weight (g)
Liver weight Absolute
(g)
Liver/body
(%)
1. DEN I6 16 16 16 14 15 I4
254 260 242 266 273 270 271
2. DEN alone
15
267 f
19
3. Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon Dicloran
10 9 10 8 10 10 10
268 286 254 284 282 288 274
I6 IO 9 20 15 12 I5
Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon Dicloran
Significantly
different
from group
2 at
f l5C f I7 f 15a f 9 f 10 ZIZ13 f 14
f f f f f f f
8.8 9.9 8.1 10.9 II.5 10.6 12.5
8.4 f 9.0 10.8 8.3 12.7 12.1 10.9 12.1
3.49 3.80 3.35 4.09 4.19 3.90 4.62
f f * f f + f
0.09a 0.45a 0.22b 0.15” 0.14” 0.18” 0.28a
1.0
3.13
l
0.16
0.8 0.6 0.6 1.1 0.5 0.5 0.6
3.36 3.77 3.28 4.47 4.30 3.79 4.42
f 0.15 l 0.14 + 0.22 f 0.22 f 0.21 f 0.22 f 0.16
f 0.6 f 1.2” f 0.7 f 0.6” l 0.7a f 0.8= f 0.9a
f f f f f f f
aP < 0.001, bP < 0.01or 'P < 0.05, respectively.
62
Table II.
Results
Group
of GST-P
Treatment
positive
foci analysis. No. of rats
GST-P positive
foci
Number
(n/cm’)
Area (mm’icm’)
16.08 12.81 6.92 13.89 12.70 18.76 6.60
4.62’ 4.07h 2.43 3.29” 2.46’ 2.94” 2.59
1.48 I.11 0.48 1.27 1.04 1.63 0.50
1. DEN Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon Dicloran 2. DEN alone 3. Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon Dicloran
16 16 16 16 14 15 14
f f f f f f f
f f + f f f f
0.49” 0.49c 0.20’ 0.39” 0.27b 0.37” 0.33
15
8.32 f 3.71
0.72 f 0.31
IO 9 IO 8 IO IO IO
0 0 0 0 0 0.22 f 0.27 0
0 0 0 0 0 0.01 f 0.02 0
GST-P positive foci larger than 0.2 mm in diameter were counted. Significantly different from group 2 at “P < 0.001, bP < 0.01 or ‘P < 0.05, respectively
without DEN initiation are summarized in Table II. The number of foci in the control group was 8.32/cm2 and the area was 0.72 mm2icm’ Vinclozolin, malathion, chlorobenzilate. tecnazene and isoproturon increased GST-P positive foci development over the control levels in both parameters. Dicloran did not affect foci development, and DEF rather decreased the area of foci. In the non-DEN-initiated groups, only isoproturon induced a small number of GST-P positive foci larger than 0.2 mm in diameter. Discussion Since this assay model is based on the two-step hypothesis of carcinogenesis and on the induction of pre-neoplastic hepatocyte lesions, it primarily provides information on whether a test compound possesses hepato-promoting activity in the rat [6]. However, we think the carcinogenic potential of chemicals can be best assessed by detection of their promoting activity based on the following concepts: (i) most carcinogens possess both tumorinitiating and tumor-promoting activities, and (ii)
carcinogenicity and organotropism of carcinogenic agents might be determined mainly by their tumor-promoting activity. Actually more than 90% of the liver carcinogens could be positively detected by this system, although assessment of carcinogens other than established hepatocarcinogens gave less positive results [3-61. In the present study, five of the seven pesticides were positive: chlorobenzilate, vinclozolin, malathion, tecnazene, and isoproturon. Dicloran was negative in terms of both the number and area of GST-P positive foci, and DEF decreased the area of foci under the present experimental conditions. Dicloran increased the liver weight and was negative in this system. DEF exerted the strongest general toxicity among the seven chemicals; it rather decreased the foci induction at 200 ppm, the lowest dose level among the seven chemicals. Thus the positivities for the pesticides examined were not directly correlated to general and/or liver toxicity of the chemicals as evaluated by the weight data, although some reports indicate that chemicals that increased liver weight enhanced the development of pre-neoplastic foci of the liver [ 181.
63
Table III.
Comparison
01‘ positivity.
Chemical
Chlor.. inated
Vinclozolin Malathion DEF Chlorobenzilate Tecnazene Isoproturon
+ -
Dicloran
+ + +
Mutagenicity
? _ ? ? -
Carcinogenicity
Liver system
-
+ +
? + ? ? ?
+ + + -
+ Positive or yes; - negative or no; ? unknown. Mutagenicity in the Ames test. References: [9-161
The results are co:mpared with their reported mutagenicity in the Ames test and reported carcinogenicity (Table III). Mutagenicity in the Ames test is negative for vinclozolin, malathion, chlorobenzilate, and dicloran, and unknown to us for the other three compounds [8-14,19,20]. Among the seven pesticides, chlorobenzilate has been reported to be carcinogenic, but only in the mouse liver [8-lo]. Similar cases of non-mutagenic carcinogens are more commonly observed for the other chlorinated compounds [ 19,201. The positive results for malathion and vinclozolin are interesting, as these two chemicals have been evaluated as ‘non-carcinogens’ in both rats and mice and non-mutagenic in the Ames test [13,19.20]. Carcinogenicity for DEF, dicloran, tecnazene and isoproturon has not 1.~11~evaluated as far as we know. Malathion has been reported as ‘noncarcinogenic’ in NTP technical reports [ 11,121. However, it is worth noting that this agent has demonstrated significant enhancement of the development of several types of tumor in both rats and mice, and the combined incidence of hepatocellular carcinomas and neoplastic nodules in male mice showed a significant linear trend when compared with either the matched controls (P = 0.041) or the pooled controls (P = 0.019) when administered at concentrations of 8000 and 16 000 ppm in the diet. Although liver carcinogenicity was not evident for malathion in either Osborne Mendel or Fischer rats, our results suggest that malathion possesses tumor-promoting activity in the liver. Vinclozolin has also been reported to be
non-carcinogenic by the FAO and WHO [13]; however, an apparent increase in liver weight was noted for both rats and mice to which the chemical was administered at concentrations of up to 4370 ppm in the diet. The level of positivity in this system was almost the same as that of phenobarbital at 0.05% in the diet. Phenobarbital has been considered a typical hepatopromoter and recently evaluated as a liver carcinogen [21]. There seems to be a discrepancy, therefore, between the reported hepatocarcinogenicity in the rat and the positivity in the present system for three chemicals. We consider, however, that these three compounds are possible hepatopromoters in the rat, and this activity has not been fully demonstrated in the long-term carcinogenicity study in this species. It is possible to conclude that chemicals exerting a positive result in this system might possess hepatopromoting activity even if the hepatocarcinogenicity has not been demonstrated in a longterm study in the rat. In the strategy for cancer prevention, we believe that a battery of rapid tests for carcinogenicity in the liver has an important role to play. Acknowledgments
The authors would like to express their gratitude to the late Dr. Kiyomi Sato, Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki, for generously providing GST-P antibody. This work was supported in part by grants in aid for cancer research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan, and grants from the Society for the Promotion of Pathology of Nagoya and the Experimental Pathological Research Association of Nagoya, Japan. Ricardo Cabral was supported by the Program for the Invitation of Foreign Scientists to Japanese Institutes by the Foundation for Promotion of Cancer Research, Tokyo, Japan. References 1
Ferber. A. and Cabral, R. (1991) Toxic epidemics caused by alimentary exposure to pesticides: a review. Food Additives Contam.. 8. 755-776.
64
6
8
International Agency for Research on Cancer (1991) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol. 53: Occupational Exposures in Insecticide Application, and Some. Pesticides. IARC Scientific Publications, Lyon. Ito, N., Tsuda, H., Tatematsu, M., Inoue, T., Tagawa. Y.. Aoki, T., Uwagawa, S., Kagawa, M., Ogiso. T.. Masui, T., Imaida, K., Fukushima, S. and Asamoto, M. (1988) Enhancing effect of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in rats: an approach for a new mediumterm bioassay system. Carcinogenesis, 9, 387-394. Hasegawa, R. and Ito, N. (1992) Liver medium-term bioassay in rats for screening of carcinogens and modifying factors in hepatocarcinogenesis. Food Chem. Toxicol., 30, 979-992. Ito, N., Shirai, T. and Hasegawa, R. (1992) Medium-term bioassay for carcinogens. In: Mechanisms of Carcinogenesis in Risk Assessment, pp. 353-388. Editors: H. D.B. McGregor and A.J. Vainio, P.N. Magee. McMichael. IARC, Lyon. Ito, N., Imaida, K., Hasegawa, R. and Tsuda, H. (1989) Rapid bioassay methods for carcinogens and modifiers of hepatocarcinogenesis. CRC Crit. Rev. Toxicol.. 19, 385-415. Cabral, R., Hoshiya, T., Hakoi, K., Hasegawa, R., Fukushima, S. and Ito, N. (1991) A rapid in vivo bioassay for the carcinogenicity of pesticides, Tumori, 77, 185-188. National Toxicology Program (1978) Toxicology and carcinogenesis studies of chlorobenzilate in rats (Technical Report Series no. 75). U.S. Department of Health and Human Services, Public Health Service, Research Triangle Park. International Agency for Research on Cancer (1983) IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans: Miscellaneous Pesticides. IARC, Lyon. International Agency for Research on Cancer (1987) IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man: Some Organochlorine Pesticides, pp. 75-81. IARC, Lyon. National Toxicology Program (1978) Toxicology and carcinogenesis studies of malathion in rats (Technical Report
12
16
I7
18
19
20
21
Series no. 24). U.S. Department of Health and Human Services, Public Health Service. Research Triangle Park. National Toxicology Program (1979) Toxicology and carcinogenesis studies of malathion in F344M rats and B6C3Ft mice (Technical Report Series no. 192). U.S. Department of Health and Human Services, Public Health Service, Research Triangle Park. FAO-WHO (1986) Pesticide Residues in Food: 1986 Evaluations. pp. 193-207. World Health Organization, Geneva. FAO-WHO (1977) Pesticide Residues in Food: Report. 1977, pp. 24-26. World Health Organization, Geneva. Tatematsu, M., Mera, Y., Ito. N., Satoh. K. and Sato. K. (1985) Relative merits of immunohistochemical demonstration of placental A, B and C forms of glutathione Stransferase and histochemical demonstration of yglutamyl transferase as markers of altered foci during liver carcinogenesis in rats. Carcinogenesis. 6, 162 1- 1626. Tatematsu, M., Takano, T., Hasegawa, R.. Imaida, K., Nakanowatari, J. and Ito, N. (1980) A sequential quantitative study of the reversibility or irreversibility of liver hyperplastic nodules in rats exposed to hepatocarcinogens. Gann, 71, 843-1755. Gad, S.C and Weil, C.S. (1982) Statistics for Toxicologists. In: Principles and Methods of Toxicology. pp. 273-320. Editor: A.W. Hayes. Raven Press, New York. Tatematsu, M.. Nakanishi, K., Murasaki, C., Miyata. Y., Hirose, M. and Ito. N. (1979) Enhancing effect of inducers of liver microsomal enzymes on induction of hyperplastic liver nodules by N-2-fluorenylacetamide in rats. J. Nat. Cancer Inst.. 63, 1411-1416. Zeiger, E. (1987) Carcinogenicity of mutagens: predictive capability of the Sulmone/lu mutagenesis assay for rodent carcinogenicity. Cancer Res., 47. I287- 1296. Ashby, J. and Tennant, R.W. (1991) Definitive relationship among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP. Mutat. Res.. 257. 229-306. International Agency for Research on Cancer (1987) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Overall Evaluations of Carcinogenicity: an Updating of IARC Monographs Vols. 1 to 42, Suppl. 7. IARC Scientific Publications, Lyon.