* Division of Pathology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan;
Department of Molecular and Environmental Pathology, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan;
Laboratory of Veterinary Pathology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan; and
Panapharm Laboratories Co., Ltd., 1285, Kurisaki-machi, Uto, Kumamoto 869-0425, Japan
Received November 5, 2002; accepted January 31, 2003
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ABSTRACT |
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Key Words: kojic acid; hepatocarcinogenesis; heterozygous p53-deficient mice.
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INTRODUCTION |
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Recently, thyroid follicular cell adenomas were found to develop in B6C3F1 mice fed diet containing 1.5 or 3% KA for 20 months (Fujimoto et al., 1998). Proliferative lesions of thyroid follicular cells were also increased in F344 rats fed diet containing 2 or 4 % KA for 12 weeks after initiation with N-bis(2-hydroxypropyl)nitrosamine [DHPN] (Mitsumori et al., 1999
; Tamura et al., 1999b
), associated with a decline in serum thyroxine (T4) and 3,5,3'-triiodothyronine (T3) and elevation of thyroid stimulating hormone (TSH) levels (Mitsumori et al., 1999
). Moreover, it has been demonstrated that KA inhibits thyroid iodine uptake and its organification (Fujimoto et al., 1999
; Tamura et al., 1999a
). Based on the findings, prolonged TSH secretion from the pituitary in response to decreased circulating thyroid hormones due to the administration of KA is presumably responsible for the thyroid tumor induction, as established for other goitrogenic substances (Hill et al., 1998
; McClain, 1992
). On the other hand, female B6C3F1 mice receiving 3% KA in the diet were also found to develop hepatocellular tumors in the first experiment conducted by Fujimoto et al.(1998)
, but consistent interpretation or underlying mechanisms of liver tumorigenicity remain uncertain. A number of in vitro mutagenicity assays have been performed for KA and the Ames test using Salmonella strains revealed positive results in the presence and absence of rat S9-mix fraction (Shibuya et al., 1982
; Wei et al., 1991
). In addition, positive results were obtained for sister chromatid exchange and chromosomal aberrations in Chinese hamster ovary cells (Wei et al., 1991
), but not in the forward gene mutation assay in Chinese hamster lung V79 cells (Shibuya et al., 1982
) in vitro. However, data for genotoxicity of KA in vivo are limited, and a negative result was obtained in a dominant lethal test (Shibuya et al., 1982
). It has been reported that heterozygous p53-deficient mice, in which one allele of the p53 gene is inactivated, derived from C57BL/6 or CBA origins provide a useful model to detect carcinogens, genotoxic carcinogens in particular, within a short-term treatment period of less than six months (Dunnick et al., 1997
; Mitsumori et al., 2000
; Ozaki et al., 1998
). Spontaneous tumor development during short-term assays is generally rare although a low incidence of lymphomas and lung adenomas or adenocarcinomas is known in both strains of p53 hemi-knockout mice (Mahler et al., 1998
, Takizawa et al., 2001
). A number of tissues or organs are counted as target for carcinogen in p53(+/-) mice. These include vascular tissue (Carmichael et al., 2000
), lymphatic organs (Dunnick et al., 1997
), urinary bladder (Ozaki et al., 1998
; Tennant et al., 1996
), uterus (Mitsumori et al., 2000
), and liver (French et al., 2001
). In contrast, p53 hemi-knockout mice are reported to not respond to nongenotoxic carcinogens that can induce tumors in the liver or kidney in conventional long-term assays (Spalding et al., 2000
). Heterozygous p53-deficient CBA mice were also reported to develop proliferative lesions in response to a single administration of N-dimethylnitrosamine (Onodera et al., 2001
). It is possible that KA exerts carcinogenic potential via genotoxic effects in the liver or other organs, and the present study was performed using heterozygous p53-deficient CBA mice and wild-type littermates in order to further evaluate this question.
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MATERIALS AND METHODS |
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Experimental animals.
The mice used in the present study, heterozygous p53-deficient CBA mice [p53(+/-) mice] in which exon 2 of the lateral p53 allele was inactivated, were F1 offspring of heterozygous p53-deficient C57BL/6J male mice back-crossed with CBA female mice (Tsukada et al., 1993). Twenty-seven male p53(+/-) mice and 35 male wild-type littermates [p53(+/+) mice], six or seven weeks of age, were purchased from Oriental Yeast Co. and used after an acclimatization period of one week. They were housed at a maximum of five per cage in plastic cages with soft chip bedding in a room that was controlled for light-dark cycle (1212 h, lights on 07001900 h), ventilation (air-exchange rate of 18 times per h), temperature (2125°C), and relative humidity (5060%). The cages and the chip bedding were exchanged for new ones twice a week. Each animal had free access to basal diet and tap water. The experiment was carried out in accordance with the Guide for Animal Experimentation of the National Institute of Health Sciences of Japan.
Experimental design.
p53(+/-) mice were randomly allocated into one group of seven animals and two groups of 10 animals based on their body weights. p53(+/+) mice were allocated in a similar manner into three groups consisting of 10, 12, and 13 animals. Groups of p53(+/-) and p53(+/+) mice were fed the diet containing 1.5 or 3% KA for 26 weeks, and the remaining group, serving as a control, was fed basal diet for the same period. During the study period, the animals were observed once a day for their general condition, and weighed once weekly.
At the end of the treatment period, surviving animals were sacrificed after blood sampling for hormone assays under deep ether anesthesia, and autopsied. Livers and thyroids were dissected out and their weights were recorded. These organs and the pituitary, spleen, lungs, and other tissues or organs with macroscopic lesions were fixed in 10% neutral buffered formalin. Next, all lobes of the liver and other organs and tissues were processed routinely, embedded in paraffin, sectioned at 45 µm, and stained with hematoxylin and eosin for histopathological examination. In addition, tissue sections were immunohistochemically stained for proliferating cell nuclear antigen (PCNA) using an anti-PCNA mouse monoclonal antibody (DAKO, Glostrup, Denmark) at a dilution of 1:100 with a blocking reagent (Vector MOMTM, Vector Labs, Inc., Burlingame, CA) and an avidin-biotin peroxidase complex kit (DAKO) with 3, 3'-diaminobenzidine as the chromogen followed by counterstaining with hematoxylin. Five thousand hepatocellular nuclei in normal background parenchyma in each animal were counted for determination of PCNA positivity.
Hormone assays.
Blood samples were obtained from the abdominal aorta for determination of serum T3, T4, and TSH levels. For this purpose, coat-A-Count Canine T3 and DPC total T4 assay kits (Diagnostic Products Corporation, Los Angeles, CA) and a rat thyroid stimulating hormone [125I] assay system (Amersham Pharmacis Biotech, Buckinghamshire, U.K.) were employed.
Statistical analysis.
Body weights, organ weights, serum hormone levels, and multiplicities of proliferative lesions were analyzed by one-way analysis of variance for homogeneity followed by Dunnett's test for comparison with the 0% KA control group. PCNA-positivity was analyzed by a nonparametric Dunnett's type test. Incidences of proliferative lesions observed were analyzed with the Fisher's exact test. The dose-response-relationship was analyzed with the Cochran Armitage test for histopathological findings and by Jonckheer's trend test for other data. Incidences of histopathological findings and PCNA-positivity were compared between p53(+/-) and p53(+/+) mice using the Fischer's exact test and Wilcoxson rank-sum test, respectively. Significance was inferred at either 5 or 1% levels.
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RESULTS |
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DISCUSSION |
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In the present experiment, serum T4 levels in mice receiving KA were reduced in a dose-related manner, but dose-proportional effects on T3 or TSH were not observed. Similar inconsistent alteration of thyroid hormones or TSH was also reported in F344 rats receiving KA at dietary concentrations up to 2% for 20 weeks (Tamura et al., 2001) and B6C3F1 mice given 1.5 or 3% KA for 20 months (Fujimoto et al., 1998
). Moreover, in our previous study in rats, administration of sulfadimethoxine or thiourea, a goitrogenic anti-thyroidal compound, was associated with a reduction of thyroid hormones and elevation of TSH after a one-week treatment but no apparent alteration of T3, T4, or TSH was evident at 19 weeks (Onodera et al., 1994
). Hood et al.(1999)
also reported only transient alterations of serum T3, T4, and/or TSH levels on prolonged administration to rats of phenobarbital, propylthiouracil or pregnenolone-16
-carbonitrile. These findings suggest that hormonal desensitization may be induced by prolonged anti-thyroidal treatments (Shimo et al., 1994
; Wynford-Thomas et al., 1982
). KA is reported to interfere with thyroid iodine uptake and its organification (Fujimoto et al., 1999
; Tamura et al., 1999a
) but not elicit any changes in the activity of hepatic uridine diphosphate glucuronosyl transferase or histopathological hypertrophy or swelling of hepatocytes in F344 rats (Mitsumori et al., 1999
). There was also no hepatocellular hypertrophy in the present study, although the KA-treated animals showed somewhat elevated liver weights. Considering the results, KA might exert goitrogenic action via hormonal mechanisms in p53(+/-) and p53(+/+) mice of CBA-background, as observed in B6C3F1 mice and F344 rats. The fact that KA failed to form DNA adducts in the thyroid glands of rats by dietary feeding at 2% (unpublished data) supports our inference.
Hepatocellular adenomas as well as altered hepatocellular foci were observed in KA-treated groups not only in p53(+/-) mice but also in their wild-type littermates. Since no spontaneous hepatocellular proliferative lesions were observed in control animals in line with the previous 26-week studies (Mitsumori et al., 2000; Onodera et al., 2001
; Takizawa et al., 2001
), these proliferative lesions in the liver could be attributed to the treatment with KA. The fact that focal hepatocellular necrosis and inflammatory cell infiltration were enhanced in the 1.5 and 3% KA groups suggests a hepatotoxic potential of KA. The elevated proliferation indices for hepatocytes were roughly parallel the occurrence of necrotic lesions both in p53(+/-) and p53(+/+) mice, and might make the large variability in the p53(+/+) mice. Although the effects of KA were masked by the relatively variable control level of the 0% KA group in p53(+/+) mice, the elevated proliferation index in the 3% group of p53(+/-) mice might also be indicative of the hepatic regeneration. In the 20-month carcinogenicity study conducted by Fujimoto et al.(1998)
, a slight (10%) but significant increase in the incidence of hepatocellular carcinomas was observed in female B6C3F1 (C57BL x C3H/He) mice receiving 3% KA in the diet, but no hepatic disorders were observed on histopathological examination as well as serum biochemistry. The findings suggest a high susceptibility of CBA-background mice particularly p53(+/-) mice to hepatotoxicity of KA, and thus associated secondary cell proliferation might influence the induction of hepatocellular tumors. In addition, significant tumorigenic dose was lowered and the prevalence of hepatic proliferative lesions was higher in the p53(+/-) mice as compared to their wild-type counterparts. In particular, incidences of hepatic tumors at a dose of 1.5% and altered foci at a dose of 3% KA in p53(+/-) mice were significantly higher than in p53(+/+) mice. Since p53(+/-) mice are sensitive to genotoxic carcinogens (Mitsumori et al., 2000
; Tennant et al., 1995
, 1996
), the possibility that KA exerts carcinogenic action through genotoxicity cannot be ruled out. With respect to the genotoxicity of KA in vitro, there have been positive results reported in the reverse gene mutation assay (Ames test) using microorganisms in the presence or absence of rat S-9 mix fraction (Shibuya et al., 1982
; Wei et al., 1991
). KA was also reported to induce DNA breaks and a clastogenic effect in cultured rat liver cells (Kinoshita et al., 1968
; Stark, 1980
) as well as sister chromatid exchange and chromosomal aberration in Chinese hamster ovary cells (Wei et al., 1991
). However, KA failed to induce positive response in dominant lethal test in vivo. From these facts, mutagenic potential of KA in vitro is indicated, but in vivo genotoxicity and mechanisms of action of liver tumorigenicity remain to be clarified. Further investigations focusing on induction or promotion activity of KA in the liver as well as in vivo genotoxicity toward hepatocytes need to be performed.
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ACKNOWLEDGMENTS |
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NOTES |
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