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Inhibitory potential of acetaminophen and o-, m-, p-aminophenols for development of γ-glutamyltranspeptidase-positive liver cell foci in rats pretreated with diethylnitrosamine
19
Cancer Letters, 28 (1985) 19-25 Elsevier Scientific Publishers Ireland Ltd.
INHIBITORY POTENTIAL OF ACETAMINOPHEN AND o-, m-, pAMINOPHENOLS FOR DEVELOPMENT OF y-GLUTAMYLTRANSPEPTIDASE-POSITIVE LIVER CELL FOCI IN RATS PRETREATED WITH DIETHYLNITROSAMINE
YASUSHI KURATA,
HIROYUKI
TSUDA, SEIKO TAMANO and NOBUYUKI
First Department of Pafhology, Nagoya City University Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467 (Japan)
IT0
1 Kawasumi,
(Received 11 March 1985) (Accepted 6 May 1985)
SUMMARY
The inhibitory potential of treatment with acetaminophen (AAP) and 3 different isometric forms of monoaminophenols (o-, m-, p-AI’) for the development of r-glutamyltranspeptidase-positive (r-GT’ ) liver cell foci was examined in an in vivo short-term assay system. Rats were initially given a single intraperitoneal injection of diethylnitrosamine (DEN) and fed test compounds from week 2 until they were killed at week 6, all rats being subjected to two-thirds partial hepatectomy at week 3. All 4 compounds exerted obvious inhibition of the development of r-GT foci with both number and area (mm* ) being reduced. AAP proved the most potent agent whereas dose-related inhibitory potential was observed within the 2 doses of p-AP treated.
INTRODUCTION
Recent application of the 2-stage concept of chemical carcinogenesis in organs other than skin has made possible the development of models for the examination of various compounds for their ability to modulate tumorigenesis [ 2,3,8,1’7,19,24]. Liver carcinogenesis, in particular, has lent itself to the short-term detection of promoting agents using liver cell foci initiated by 2-acetylaminofluorene (2-AAF) or DEN as the end point for quantitation [2,9-11,22,26]. Furthermore, short-term tests have also been found advantageous for the assay of inhibitory agents such as butylated hydroxyanisole and nafenopin
L&201. 0304-3835/85/$03.30 0 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
20
Our laboratory has previously demonstrated that AAP (paracetamol, p-acetaminophenol), which has been used as an analgesic and antipyretic drug, clearly inhibits the development of preneoplastic and neoplastic liver of rats initially treated by N-ethyl-N-hydroxyethylnitrosamine (EHEN) [ 281. In this study, AAP, its metabolite p-AP as the isometric monoaminophenol derivatives o- or m-AP were tested for inhibitory potential on the development of y-GT’ foci in the liver of rats pretreated with DEN. MATERIALS
AND METHODS
Animals
Male, 6-week-old, F344 rats (Charles River Japan, Inc., Atsugi, Japan) were housed in plastic cages in an air-conditioned room at 24°C with a 12 h light/dark cycle. They were given tap water ad libitum. All animals were fasted for 18 h prior to killing. Chemicals
and diet
DEN, AAP, o-AAP, m-AP and p-AP were purchased from Tokyo Chemical Industry Co. Ltd., Tokyo, Japan. They were mixed into powdered basal diet (Oriental M, Oriental Yeast Co., Tokyo, Japan) at concentrations of 1.0% (AAP, o-AP and m-AP) or 1.0% and 0.5% @-AP). Experimental
protocol
A total of 275 rats were divided into 3 groups. Group 1 animals were initially given a single intraperitoneal injection of 200 mg/kg of DEN dissolved into 0.9% NaCl solution. After 2 weeks on basal diet, rats were placed on basal diet containing one of the test compounds for 6 weeks. All animals were subjected to two-thirds partial hepatectomy at week 3 and killed for examination at week 8. Group 2 was given DEN and partial hepatectomy as in group 1 but not fed test compound. Group 3 animals were injected with 0.9% NaCl solution instead of DEN solution then subjected to feeding of one test compound and also partial hepatectomy in the same manner as in group 1. Subgroups for each test chemical consisted of 25 rats. Body weight was measured weekly and the liver weight was recorded at the time of killing. Histological
examination
and quantitation
of y-GT’
foci
At the time of killing, the liver was excised and l-2 mm sections were cut with a razor blade. Four slices, one each from the right posterior and caudate lobes and 2 from the right anterior lobes, were fixed in ice-cold acetone and processed for paraffin sectioning for subsequent histological examination of y-GT’ foci as described previously [ 2,8,21,23,25]. The numbers and area of r-GT’ foci of more than 0.2 mm diameter were measured using a color video image processor (Olympus VIP-21C; Olympus-Ikegami Tsushin Co., Tokyo, Japan) and expressed as number/cm’ or area (mm’ )/cm*. The measurement was carried out without reference to the treatment group of the slides. Statistical analysis was performed using Student’s t-test.
21
RESULTS
Survival, body and liver weights Survival ratios in groups given test compounds were between 60% to 80% of controls. Animals given AAP (groups 1 and 3) suffered greatest loss. Animals subjected to feeding of test compounds with (group 1) or without (group 3) prior injection of DEN showed obvious decrease of body weight. Significant decrease was also observed when the values in group 1 were compared to those of group 2 in which rats were given DEN alone. Among the group 1 animals, those given m-AP showed the most remarkable decrease. A dose-dependent decrease in body weight was observed in the subgroups given different doses of p-AP (1.0% and 0.5%). Animals given AAP or o-AP in group 1, showing slight, but not statistically significant increase of liver weight were not statistically significant when compared to group 2. However, liver weight/body weight ratio (7%of body wt) in rats given o-AP in group 1 was significantly increased compared to group 2 (Table 1). Quantitative values of y-GT’ foci All 4 related compounds tested were associated with significantly decreased number and area of r-GT’ foci as compared to values for group 2. AAP
TABLE
1
AVERAGE Group
BODY
AND LIVER
Treatment DEN
WEIGHTS
Dosea
No. of rats
Body weightb
Test chemical
Liver weightb Average
Ratio to body weight (%I
1
+ + + +
AAP 0-AP
m-AP P-D
1.0 1.0
1.0 1.0
L -
AAP o-AP
m-AP P-M
19
1.0
17 18 17 16
1.0 1.0 1.0 0.5
18 20 16 23
0.5 2 3
15 15
244.9 225.0 219.2 225.0 242.2 258.1 268.4 245.9 265.2 251.7 265.6
a Percentage mixed in the basal diet. b Mean + S.D. ’ Significantly different from group 2 at P < 0.05. d Significantly different from group 2 at ’ Significantly different from group 2 at
P < 0.01. P < 0.001.
i * r + f + f + r k +
20.3’ 25.ge 18.7e 18.1e 19.0d 12.8 14.7 17.6 19.1 25.2 19.6
7.71 + 1.66 7.57 t 2.39 6.46 f. 1.57 7.05 k 1.48 6.94 + 1.53 7.12 * 1.39 8.89 + 1.46 8.71 f 1.87 8.94 t 1.66 8.26 f 1.40 7.64 f. 1.42
3.13 3.30 2.92 3.11 2.84 2.75 3.31 3.52 3.35 3.26 2.87
f 0.51 r 0.70c +_0.49 ?r0.43 * 0.43 r 0.43 + 0.43 ? 0.62 * 0.41 +_0.35 * 0.41
22 TABLE 2 QUANTITATIVE Group
2 3
OF r-GT’ FOCI IN THE LIVER
Treatment DEN
1
VALUES
Dosea Test chemical
t
AAP
+
0-AP
+
m-AP
+
P-M
t -
-
-
No. of rats
AAP 0-AP
-
m-AP
-
P-M
1.0 1.0 1.0 1.0 0.5 1.0 1.0 1.0 1.0 0.5
15 15 19 17 18 17 16 18 20 16 23
r-GT’ focib NoJcm’
Area (mm2 )/ cm1
3.76 3.90 5.92 4.35 6.28 9.92 0 0 0 0 0
0.19 0.26 0.33 0.25 0.35 0.53 0 0 0 0 0
k 1.84d f 1.90d t 2.06d 5 1.78d ?r1.78’ t 3.24
+ 0.12d * 0.12d f. o.loc f 0.08d k O.llC + 0.19
* Percentage mixed in the basal diet. bMean * S.D. c Significantly different from group 2 at P < 0.01. d Significantly different from group 2 at P < 0.001
caused the most remarkable decrease in both number (3.76 f 1.84 vs. 9.92 f 3.24) and area (0.19 rt 0.12 vs. 0.53 + 0.19). A dose-related decrease in the number and area of r-GT’ foci was also observed for the 2 different doses of p-AP used (0.5% and 1.0%). Treatment with the 4 compounds without initial injection of DEN (group 3) did not induce any -y-GT’ foci (Table 2). DISCUSSION
The results clearly show that AAP and related compounds having monoaminophenol chemical structure exerted inhibitory effects on the development of y-GT’ foci in the liver of rats pretreated with DEN and indicate the utility of this model for demonstrating modifying potential as demonstrated earlier for a variety of promoting agents [2,8,21,25,26]. Presumably, since the inhibitory effect is to form visible islands subsequent to initiation, the number of initiated cells should remain unchanged and only growth should be affected. Only foci greater than 0.2 mm in diameter were included in the quantitative analysis in the present experiment and, accordingly, decrease in number of foci is interpreted as representing growth-Lhibitory effect on foci [ 25,281. A similar inhibitory effect was also observed when AAP was given after initial treatment with EHEN suggesting general inhibitory potential irrespective of initiating agent. As with butylated hydroxytoluene and ethoxyquin, tested using the same protocol, AAP clearly inhibited the develop-
23
ment of not only y-GT’ foci but also hyperplastic nodules and hepatocellular carcinomas [ 271. Although p-AP is the only one of the 3 related compounds tested which is known to be a metabolite of AAP [14,16], inhibitory effect was associated with o-AP and m-AP treatment. Therefore their inhibitory effect is likely to be related to the aminophenol structure itself. AAP and p-AP were well known for their nephrotoxic effects, inducing tubular necrosis in the proximal straight segments, whereas m-AP and o-AP do not exert such influence [12,14--161. Therefore, it seems that possible systemic changes associated with nephrotoxic effects do not correlate with inhibition of development of -y-GT’ foci in the liver. AAP and p-AP are also hepatotoxic agents capable of causing centrilobular necrosis [ 1,5,12], apparently due to the formation of an active metabolite, postulated as N-acetylimidoquinone, through the action of cytochrome P-450 enzymes [1,18]. Carbon tetrachloride, which in contrast normally enhances hepatocarcinogenesis, also causes centrilobular necrosis [ 41, therefore, suggesting that the hepatotoxic effect of AAP may be irrelevant to its inhibition potential. On the other hand, the changed drug metabolizing systems known to be characteristic for initiated cells, may in an appropriate fashion to the selective toxicity of 2-AAF to non-initiated hepatocytes which led to a postulated promotion of hepatocsrcinogenesis, make them more susceptible to AAP toxicity [6]. Further studies are required to clarify the correlation between cell toxicity and promotive or inhibitory potential in the liver. The present results viewed in conjunction with the fact that AAP has been shown to enhance the induction of kidney tumors [27] and to be a hepatocarcinogen to IF mice [ 71, and phenacetin, the metabolic precursor of AAP, is known to enhance urinary bladder carcinogenesis [ 131, indicate the importance of evaluation of AAP and possible related compounds for modifying potential. The multiplicity of target organs suggests, based on the adaptation model, a whole body carcinogenesis concept with wide-spectrum multipotential carcinogen such as N-nitrosomethylurea [28]. ACKNOWLEDGEMENT
This work was supported by a Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan and by the Ministry; of Health and Welfare. REFERENCES 1 Black, M. (1980) Acetaminophen hepatotoxicity. Gastroenterology, 78, 382-392. 2 Cameron, R.G., Imaida, K., Tsuda, H. and Ito, N. (1982) Promotive effects of steroids and bile acids on hepatocarcinogenesis initiated by diethylnitrosamine. Cancer Res., 42,2426-2428.
24 3 Cohen, S.M., Arai, M., Jacobs, J.B. and Friedell, G.H. (1979) Promoting effect of saccharin and DL-tryptophan in urinary bladder carcinogenesis. Cancer Res., 39, 1207-1217. 4 Craddock, V.M. (1976) Cell proliferation and experimental liver cancer. In: Liver Cell Cancer, pp. 153-201. Editors: H.M. Cameron, D.S. Linsell and G.P. Warwick. Elsevier, Amsterdam. 5 Dixon, M.F., Nimmo, J. and Prescott, L.F. (1971) Experimental paracetamol-induced hepatic necrosis: a histopathological study. J. Pathol. Bacterial., 103, 225-229. 6 Farber, E., Parker, S. and Gruenstein, M. (1976) The resistance of putative premalignant liver cell populations, hyperplastic nodules, to the acute cytotoxic effects of some carcinogens. Cancer Res., 36, 3879-3887. 7 Flaks, A. and Flaks, B. (1983) Induction of liver cell tumors in IF mice by paracetamol. Carcinogenesis, 4, 363-368. 8 Imaida, K., Fukushima, S., Shirai, T., Ohtani, M., Nakanishi, K. and Ito, N. (1983) Promoting activities of butylated hydroxyanisole and butylated hydroxytoluene on 2-stage urinary bladder carcinogenesis and the inhibition of r-glutamyl transpeptidasepositive foci development in the liver of rats. Carcinogenesis, 4,895-899. 9 Ito, N., Tatematsu, M., Nakanishi, K., Hasegawa, R., Takano, T., Imaida, K. and Ogiso, T. (1980) The effects of various chemicals on the development of hyperplastic liver nodules in hepatectomized rats treated with N-nitrosodiethylamine or N-2fluorenylacetamide. Gann, 71, 832-842. 10 Ito, N., Tsuda, H., Hasegawa, R. and Imaida, K. (1982) Sequential observation of pathomorphologic alterations in preneoplastic lesions during the promoting stage of hepatocarcinogenesis and the development of short-term test system for hepatopromoters and hepatocarcinogens. Toxicol. Pathol., 10, 37-49. 11 Ito, N., Tsuda, H., Hasegawa, R. and Imaida, K. (1983) Comparison of the promoting effects of various agents in induction of preneoplastic lesions in rat liver. Environ. Health Perspect., 50, 131-138. 12 Mitchell, J.R., Jollow, D.J., Potter, W.Z., Davis, D.C., Gillette, J.R. and Brodie, B.B. (1973) Acetaminophen-induced hepatic necrosis, I. Role of drug metabolism. J. Pharmacol. Exp. Ther., 187, 185-194. 13 Nakanishi, K., Fukushima, S., Shibata, M., Shirai, T., Ogiso, T. and Ito, N. (1978) Effect of phenacetin and caffeine on the urinary bladder of rats treated with N-butylN-(4-hydroxybutyl)nitrosamine. Gann, 69, 395-400. 14 Newton, J.F., Kuo, C.-H., Gemborys, M.W., Mudge, G.H. and Hook, J.B. (1982) Nephrotoxicity of p-aminophenol, a metabolite of acetaminophen, in the Fischer 344 rat. Toxicol. Appl. Pharmacol., 65, 336-344. 15 Newton, J.F., Yoshimoto, M., Bernstein, J., Rush, G.F. and Hook, J.B. (1983) Acetaminophen nephrotoxicity in the rat. I. Strain differences in nephrotoxicity and metabolism. Toxicol. Appl. Pharmacol., 69, 291-306. 16 Newton, J.F., Yoshimoto, M., Bernstein, J., Rush, G.F. and Hook, J.B. (1983) Acetaminophen nephrotoxicity in the rat. II. Strain differences in nephrotoxicity and metabolism of p-aminophenol, a metabolite of acetaminophen. Toxicol. Appl. Pharmacol., 69, 307-318. 17 Peraino, C., Fry, R.J.M., Staffeldt, E. and Christopher, J.P. (1977) Enhancing effects of phenobarbitone and butylated hydroxytoluene on 2-acetylaminofluorene-induced hepatic tumorigenesis in the rat, Food Cosmet. Toxicol., 15, 93-96. 18 Potter, W.Z., Davis, D.C., Mitchell, J.R., Jollow, D.J., Gillete, J.R. and Brodie, B.B. (1973) Acetam!nophen-induced hepatic necrosis. III. Cytochrome P-450-mediated convalent binding in uitro. J. Pharmacol. Exp. Ther., 187, 203-210. 19 Reddy, B.S., \,dtanabe, K. Weisburger, J.H. and Wynder, E.L. (1977) Promoting effect of bile acids in colon carcinogenesis in germ-free and conventional F344 rats. Cancer Res., 37, 3238-3242.
25
20 Staubi, W., Bentley, P., Bieri, F., Froblich, E. and Waechter, F. (1984) Inhibitory effect of nafenopin upon the development of diethylnitrosamine-induced enzymealtered foci within the rat liver. Carcinogenesis, 5,41-46. 21 Tamano, S., Tsuda, H., Fukushima, S., Masui, T., Hosoda, K. and Ito, N. (1983) Dose and sex dependent effects of 2-acetylaminofluorene, 3’-methyl-4-dimethylaminoazobenzene and DL-ethionine in induction of r-glutamyl transpeptidase-positive liver cell foci in rats. Cancer Letters., 20, 313-321. 22 Tatematsu, M., Hasegawa, R., Imaida, K., Tsuda, H. and Ito, N. (1983) Survey of various chemicals for initiating and promoting activities in a short-term in uivo system based on generation of hyperplastic liver nodules in rats. Carcinogenesis, 4, 381-386. 23 Tamano, S., Tsuda, H., Tatematsu, M., Hasegawa, R., Imaida, K. and Ito, N. (1981) Induction of r-glutamyl transpeptidase positive foci in rat liver by pyrolysis products of amino acids. Gann, 12, 147-753. 24 Tsuda, H., Fukushima, S., Imaida, K., Kurata, Y. and Ito, N. (1983) Organ-specific promoting effect of phenobarbital and saccharin in induction of thyroid, liver, and urinary bladder tumors in rats after initiation with N-nitrosomethylurea. Cancer Res., 43,3292-3296. 25 Tsuda, H., Hasegawa, R., Imaida, K., Masui, T., Moore, M. and Ito, N. (1984) Modifying potential of thirty-one chemicals on the short-term development of r-glutamyl transpeptidase-positive foci in diethylnitrosamine-initiated rat liver. Gann, 75, 876883. 26 Tsuda, H., Masui, T., Imaida, K., Fukushima, S. and Ito, N. (1984) Promotive effect of primary and secondary bile acids on the induction of y-glutamyl transpeptidasepositive liver cell foci as a possihle endogenous factor for hepatocarcinogenesis in rats. Gann, 75, 871-875. 27 Tsuda, H., Sakata, T., Masui, T., Imaida, K. and Ito, N. (1984) Modifying effects of butylated hydroxyanisole, ethoxyquin and acetaminophen on induction of neoplastic lesions in rat liver and kidney initiated by N-ethyl-N-hydroxyethylnitrosamine. Carcinogenesis, 5, 525-531. 28 Tsuda, H., Sakata, T., Shirai, T., Kurata, Y., Tamano, S. and Ito, N. (1984) Modification of N-methyl-N-nitrosourea initiated carcinogenesis in the rat by subsequent treatment with antioxidants, phenobarbital and ethinyl estradiol. Cancer Letters, 24, 19-27.
Cancer Letters, 28 (1985) 19-25 Elsevier Scientific Publishers Ireland Ltd.
INHIBITORY POTENTIAL OF ACETAMINOPHEN AND o-, m-, pAMINOPHENOLS FOR DEVELOPMENT OF y-GLUTAMYLTRANSPEPTIDASE-POSITIVE LIVER CELL FOCI IN RATS PRETREATED WITH DIETHYLNITROSAMINE
YASUSHI KURATA,
HIROYUKI
TSUDA, SEIKO TAMANO and NOBUYUKI
First Department of Pafhology, Nagoya City University Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467 (Japan)
IT0
1 Kawasumi,
(Received 11 March 1985) (Accepted 6 May 1985)
SUMMARY
The inhibitory potential of treatment with acetaminophen (AAP) and 3 different isometric forms of monoaminophenols (o-, m-, p-AI’) for the development of r-glutamyltranspeptidase-positive (r-GT’ ) liver cell foci was examined in an in vivo short-term assay system. Rats were initially given a single intraperitoneal injection of diethylnitrosamine (DEN) and fed test compounds from week 2 until they were killed at week 6, all rats being subjected to two-thirds partial hepatectomy at week 3. All 4 compounds exerted obvious inhibition of the development of r-GT foci with both number and area (mm* ) being reduced. AAP proved the most potent agent whereas dose-related inhibitory potential was observed within the 2 doses of p-AP treated.
INTRODUCTION
Recent application of the 2-stage concept of chemical carcinogenesis in organs other than skin has made possible the development of models for the examination of various compounds for their ability to modulate tumorigenesis [ 2,3,8,1’7,19,24]. Liver carcinogenesis, in particular, has lent itself to the short-term detection of promoting agents using liver cell foci initiated by 2-acetylaminofluorene (2-AAF) or DEN as the end point for quantitation [2,9-11,22,26]. Furthermore, short-term tests have also been found advantageous for the assay of inhibitory agents such as butylated hydroxyanisole and nafenopin
L&201. 0304-3835/85/$03.30 0 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
20
Our laboratory has previously demonstrated that AAP (paracetamol, p-acetaminophenol), which has been used as an analgesic and antipyretic drug, clearly inhibits the development of preneoplastic and neoplastic liver of rats initially treated by N-ethyl-N-hydroxyethylnitrosamine (EHEN) [ 281. In this study, AAP, its metabolite p-AP as the isometric monoaminophenol derivatives o- or m-AP were tested for inhibitory potential on the development of y-GT’ foci in the liver of rats pretreated with DEN. MATERIALS
AND METHODS
Animals
Male, 6-week-old, F344 rats (Charles River Japan, Inc., Atsugi, Japan) were housed in plastic cages in an air-conditioned room at 24°C with a 12 h light/dark cycle. They were given tap water ad libitum. All animals were fasted for 18 h prior to killing. Chemicals
and diet
DEN, AAP, o-AAP, m-AP and p-AP were purchased from Tokyo Chemical Industry Co. Ltd., Tokyo, Japan. They were mixed into powdered basal diet (Oriental M, Oriental Yeast Co., Tokyo, Japan) at concentrations of 1.0% (AAP, o-AP and m-AP) or 1.0% and 0.5% @-AP). Experimental
protocol
A total of 275 rats were divided into 3 groups. Group 1 animals were initially given a single intraperitoneal injection of 200 mg/kg of DEN dissolved into 0.9% NaCl solution. After 2 weeks on basal diet, rats were placed on basal diet containing one of the test compounds for 6 weeks. All animals were subjected to two-thirds partial hepatectomy at week 3 and killed for examination at week 8. Group 2 was given DEN and partial hepatectomy as in group 1 but not fed test compound. Group 3 animals were injected with 0.9% NaCl solution instead of DEN solution then subjected to feeding of one test compound and also partial hepatectomy in the same manner as in group 1. Subgroups for each test chemical consisted of 25 rats. Body weight was measured weekly and the liver weight was recorded at the time of killing. Histological
examination
and quantitation
of y-GT’
foci
At the time of killing, the liver was excised and l-2 mm sections were cut with a razor blade. Four slices, one each from the right posterior and caudate lobes and 2 from the right anterior lobes, were fixed in ice-cold acetone and processed for paraffin sectioning for subsequent histological examination of y-GT’ foci as described previously [ 2,8,21,23,25]. The numbers and area of r-GT’ foci of more than 0.2 mm diameter were measured using a color video image processor (Olympus VIP-21C; Olympus-Ikegami Tsushin Co., Tokyo, Japan) and expressed as number/cm’ or area (mm’ )/cm*. The measurement was carried out without reference to the treatment group of the slides. Statistical analysis was performed using Student’s t-test.
21
RESULTS
Survival, body and liver weights Survival ratios in groups given test compounds were between 60% to 80% of controls. Animals given AAP (groups 1 and 3) suffered greatest loss. Animals subjected to feeding of test compounds with (group 1) or without (group 3) prior injection of DEN showed obvious decrease of body weight. Significant decrease was also observed when the values in group 1 were compared to those of group 2 in which rats were given DEN alone. Among the group 1 animals, those given m-AP showed the most remarkable decrease. A dose-dependent decrease in body weight was observed in the subgroups given different doses of p-AP (1.0% and 0.5%). Animals given AAP or o-AP in group 1, showing slight, but not statistically significant increase of liver weight were not statistically significant when compared to group 2. However, liver weight/body weight ratio (7%of body wt) in rats given o-AP in group 1 was significantly increased compared to group 2 (Table 1). Quantitative values of y-GT’ foci All 4 related compounds tested were associated with significantly decreased number and area of r-GT’ foci as compared to values for group 2. AAP
TABLE
1
AVERAGE Group
BODY
AND LIVER
Treatment DEN
WEIGHTS
Dosea
No. of rats
Body weightb
Test chemical
Liver weightb Average
Ratio to body weight (%I
1
+ + + +
AAP 0-AP
m-AP P-D
1.0 1.0
1.0 1.0
L -
AAP o-AP
m-AP P-M
19
1.0
17 18 17 16
1.0 1.0 1.0 0.5
18 20 16 23
0.5 2 3
15 15
244.9 225.0 219.2 225.0 242.2 258.1 268.4 245.9 265.2 251.7 265.6
a Percentage mixed in the basal diet. b Mean + S.D. ’ Significantly different from group 2 at P < 0.05. d Significantly different from group 2 at ’ Significantly different from group 2 at
P < 0.01. P < 0.001.
i * r + f + f + r k +
20.3’ 25.ge 18.7e 18.1e 19.0d 12.8 14.7 17.6 19.1 25.2 19.6
7.71 + 1.66 7.57 t 2.39 6.46 f. 1.57 7.05 k 1.48 6.94 + 1.53 7.12 * 1.39 8.89 + 1.46 8.71 f 1.87 8.94 t 1.66 8.26 f 1.40 7.64 f. 1.42
3.13 3.30 2.92 3.11 2.84 2.75 3.31 3.52 3.35 3.26 2.87
f 0.51 r 0.70c +_0.49 ?r0.43 * 0.43 r 0.43 + 0.43 ? 0.62 * 0.41 +_0.35 * 0.41
22 TABLE 2 QUANTITATIVE Group
2 3
OF r-GT’ FOCI IN THE LIVER
Treatment DEN
1
VALUES
Dosea Test chemical
t
AAP
+
0-AP
+
m-AP
+
P-M
t -
-
-
No. of rats
AAP 0-AP
-
m-AP
-
P-M
1.0 1.0 1.0 1.0 0.5 1.0 1.0 1.0 1.0 0.5
15 15 19 17 18 17 16 18 20 16 23
r-GT’ focib NoJcm’
Area (mm2 )/ cm1
3.76 3.90 5.92 4.35 6.28 9.92 0 0 0 0 0
0.19 0.26 0.33 0.25 0.35 0.53 0 0 0 0 0
k 1.84d f 1.90d t 2.06d 5 1.78d ?r1.78’ t 3.24
+ 0.12d * 0.12d f. o.loc f 0.08d k O.llC + 0.19
* Percentage mixed in the basal diet. bMean * S.D. c Significantly different from group 2 at P < 0.01. d Significantly different from group 2 at P < 0.001
caused the most remarkable decrease in both number (3.76 f 1.84 vs. 9.92 f 3.24) and area (0.19 rt 0.12 vs. 0.53 + 0.19). A dose-related decrease in the number and area of r-GT’ foci was also observed for the 2 different doses of p-AP used (0.5% and 1.0%). Treatment with the 4 compounds without initial injection of DEN (group 3) did not induce any -y-GT’ foci (Table 2). DISCUSSION
The results clearly show that AAP and related compounds having monoaminophenol chemical structure exerted inhibitory effects on the development of y-GT’ foci in the liver of rats pretreated with DEN and indicate the utility of this model for demonstrating modifying potential as demonstrated earlier for a variety of promoting agents [2,8,21,25,26]. Presumably, since the inhibitory effect is to form visible islands subsequent to initiation, the number of initiated cells should remain unchanged and only growth should be affected. Only foci greater than 0.2 mm in diameter were included in the quantitative analysis in the present experiment and, accordingly, decrease in number of foci is interpreted as representing growth-Lhibitory effect on foci [ 25,281. A similar inhibitory effect was also observed when AAP was given after initial treatment with EHEN suggesting general inhibitory potential irrespective of initiating agent. As with butylated hydroxytoluene and ethoxyquin, tested using the same protocol, AAP clearly inhibited the develop-
23
ment of not only y-GT’ foci but also hyperplastic nodules and hepatocellular carcinomas [ 271. Although p-AP is the only one of the 3 related compounds tested which is known to be a metabolite of AAP [14,16], inhibitory effect was associated with o-AP and m-AP treatment. Therefore their inhibitory effect is likely to be related to the aminophenol structure itself. AAP and p-AP were well known for their nephrotoxic effects, inducing tubular necrosis in the proximal straight segments, whereas m-AP and o-AP do not exert such influence [12,14--161. Therefore, it seems that possible systemic changes associated with nephrotoxic effects do not correlate with inhibition of development of -y-GT’ foci in the liver. AAP and p-AP are also hepatotoxic agents capable of causing centrilobular necrosis [ 1,5,12], apparently due to the formation of an active metabolite, postulated as N-acetylimidoquinone, through the action of cytochrome P-450 enzymes [1,18]. Carbon tetrachloride, which in contrast normally enhances hepatocarcinogenesis, also causes centrilobular necrosis [ 41, therefore, suggesting that the hepatotoxic effect of AAP may be irrelevant to its inhibition potential. On the other hand, the changed drug metabolizing systems known to be characteristic for initiated cells, may in an appropriate fashion to the selective toxicity of 2-AAF to non-initiated hepatocytes which led to a postulated promotion of hepatocsrcinogenesis, make them more susceptible to AAP toxicity [6]. Further studies are required to clarify the correlation between cell toxicity and promotive or inhibitory potential in the liver. The present results viewed in conjunction with the fact that AAP has been shown to enhance the induction of kidney tumors [27] and to be a hepatocarcinogen to IF mice [ 71, and phenacetin, the metabolic precursor of AAP, is known to enhance urinary bladder carcinogenesis [ 131, indicate the importance of evaluation of AAP and possible related compounds for modifying potential. The multiplicity of target organs suggests, based on the adaptation model, a whole body carcinogenesis concept with wide-spectrum multipotential carcinogen such as N-nitrosomethylurea [28]. ACKNOWLEDGEMENT
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