Promoting effects of kojic acid due to serum TSH elevation resulting from reduced serum thyroid hormone levels on development of thyroid proliferative lesions in rats initiated with N-bis(2-hydroxypropyl)nitrosamine

Kunitoshi Mitsumori3, Hiroshi Onodera, Masakazu Takahashi1, Takushi Funakoshi2, Toru Tamura, Kazuo Yasuhara, Kiyoshi Takegawa and Michihito Takahashi

Division of Pathology, National Institute of Health Sciences, 1–18–1, Kamiyoga, Setagaya-ku, Tokyo 158,
1 Department of Pathology, Sasaki Institute, 2–2, Kanda Surugadai, Chiyoda-ku, Tokyo 101 and
2 Pharmacokinetics and Analysis Research, Yoshitomi Pharmaceutical Industries Ltd., 955 Koiwai, Yoshitomi-cho, Chikujo-gun, Fukuoka 871, Japan


    Abstract
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In order to examine whether kojic acid (KA) exerts a promoting effect on thyroid carcinogenesis, male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine (BHP; 2800 mg/kg body wt, single s.c. injection) and, starting 1 week later, received pulverized basal diet containing 2 or 0% KA for 12 weeks. Untreated control rats were given basal diet for 13 weeks. As an additional experiment, two groups without BHP initiation received basal diet or diet containing 2% KA for 20 weeks. The serum triiodothyronine (T3) and thyroxine (T4) levels were significantly decreased (half to one-third of values of the BHP alone group) and serum thyroid-stimulating hormone (TSH) was markedly increased (13–19 times higher than the values of the BHP-alone group) in the BHP + KA group at weeks 4 and 12. Similar changes in serum thyroid-related hormones were observed in the group with 2% KA alone at week 4, but not at week 20. Thyroid weights were significantly increased in the BHP + KA and KA-alone groups. Focal thyroid follicular hyperplasias and adenomas were observed in 4/5 and 3/5 rats in the BHP + KA group at week 4, respectively. At weeks 12, these lesions were observed in all rats in the BHP + KA group. Animals of the KA alone group showed marked diffuse hypertrophy of follicular epithelial cells at weeks 4 and 20. No changes in thyroid-related hormone levels or thyroid histopathological lesions were observed in either the BHP alone or the untreated control groups. Measurement of liver T4-uridine diphosphate glucuronosyltransferase (UDP-GT) activity at week 4 revealed no significant intergroup differences. These results suggest that thyroid proliferative lesions were induced by KA administration due to continuous serum TSH stimulation through the negative feedback mechanism of the pituitary–thyroid axis, with decreases of T3 and T4 caused by a mechanism independent of T4-UDP-GT activity.

Abbreviations: BHP, N-bis(2-hydroxypropyl) nitrosamine; KA, kojic acid; PB, phenobarbital; PPO, polyphenoloxidase; PTU, propylthiouracil; T3, triiodothyronine; T4, thyroxine; TLC, thin-layer chromatography; TSH, thyroid-stimulating hormone; TU, thiourea; UDP-GT, T4-uridine diphosphate glucuronosyltransferase.

Kojic acid [KA; 5-hydroxy-2-(hydroxymethyl)-4-pyrone] (Figure 1Go) is a secondary fungal metabolite (1,2) commonly produced by many species of Aspergillus, Acetobactor and Penicillium (3). The Aspergillus flavus group has traditionally been used in Japan for the production of fermented foodstuffs such as `miso', soy sauce and `sake'. Aspergillus oryzae produces KA with high efficiency (10 mg/ml/10 days) in the culture medium at 30°C (4). KA has bacteriostatic activity (5), and it inhibits mushroom tyrosinase (6) and polyphenoloxidase (PPO) purified from potato and white shrimp (7). Based on these effects, it has been used as an inhibitor of tyrosinase in foods and as a food additive to prevent enzymatic browning of raw crabs and shrimps due to PPO.



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Fig. 1. Structure of kojic acid [5-hydroxy-2-(hydroxymethyl)-4-pyrone].

 
KA causes DNA breaks and clastogenic effects in cultured rat liver cells (8,9), and mutations in Salmonella typhimurium (1012). It induces sister chromatid exchange and chromosomal aberrations in Chinese hamster ovary cells in the presence or absence of rat liver S9 (13). Recently, Fujimoto et al. reported the development of thyroid follicular cell adenomas in male and female B6C3F1 mice given diet containing 1.5 and 3% KA for 20 months (14). The present study was performed to assess whether thyroid tumors are similarly induced in rats treated or not treated with a known thyroid carcinogen and to determine the involvement of a non-genotoxic mechanism featuring decreased serum triiodothyronine (T3) levels and increased serum thyroid-stimulating hormone (TSH).

Male F344 rats, 5 weeks old, were obtained from Charles River (Atugi, Japan) and housed five to a polycarbonated cage with white chips as bedding in an air-conditioned room (room temperature, 23 ± 2°C; relative humidity, 60 ± 5°C; a 12 h light/dark cycle). Powdered basal diet (CRF-1, Oriental Yeast, Tokyo, Japan) and tap water were provided ad libitum. KA was obtained from Nagase Biochemical Co. (Tokyo, Japan) and N-bis(2-hydroxypropyl) nitrosamine (BHP) was purchased from Nacalai Tesque (Kyoto, Japan). After a 1 week acclimatization period, animals without any abnormal findings were selected for the present study. A total of 28 rats were used in experiment 1. Eight rats were allocated to group 1 and 10 rats each to groups 2 and 3. The initial mean body weights of each group were similar. Rats in groups 2 and 3 received a single s.c. injection of 2800 mg/kg BHP. From 1 week after the BHP initiation, the rats in group 3 were given basal diet containing 2% KA and the rats in groups 1 and 2 received basal diet alone for 12 weeks. In experiment 2, two groups of 10 rats without BHP initiation were given basal diet or basal diet containing 2% KA for 20 weeks. After measurement of body weights, blood was collected from the abdominal aorta of all animals under ether anesthesia for hormone assays followed by autopsy. Half of the rats in each group of experiment 1 were killed at week 4 and the remainder after 12 weeks exposure. Half of the rats per group of experiment 2 were killed at weeks 4 and 20.

Serum T3 and thyroxine (T4) were measured by Micro Particle Enzyme and Fluorescence Polarization Immunoassays, using a T3- and a T4-Dainapack (Abbott Laboratories, North Chicago, IL), and serum TSH with an NIADDK radioimmunoassay kit (RP-2) supplied by Dr A.F.Parlow (Pituitary and Antisera Center, Harbor-UCLA Medical Center, CA).

Determination of T4-uridine diphosphate glucuronosyltransferase (UDP-GT) activities was conducted at week 4 in experiment 1. Microsomes were prepared by the method of Kato (15). The protein concentration in the microsomes was measured by the method of Lowry et al. (16). UDP-GT activity was determined by measuring the production of T4-glucuronide as previously described (17) with minor modification. The reaction mixture (100 µl) contained 5 µM 125I-labeled T4 (14.1 kBq; New England Nuclear, Boston, MA), 2 mM uridine diphosphate glucuronic acid (UDPGA; Nacalai Tesque, Kyoto, Japan), 5 mM MgCl2, 0.05% Triton X-100 (Nacalai Tesque, Kyoto, Japan) and microsomal suspension (1.3–1.9 mg protein) in 50 mM Tris–HCl buffer (pH 7.4). After 60 min of incubation at 37°C, the incubation was terminated by methanol and centrifuged and the resultant supernatant was analyzed by thin-layer chromatography (TLC) using butanol (18). The radioactivity on the plates corresponding to T4 and T4-glucuronide was determined with a TLC-linear analyzer (Berthold, Wildbad, Germany).

At autopsy the organ weights of the thyroids, pituitary and liver were recorded. Thyroid and pituitary tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4–5 µm, and stained with hematoxylin and eosin for microscopic examination.

Quantitative data are given as mean and standard deviation, and differences were analyzed by Student's t-test. Incidences of thyroid histopathological lesions were analyzed with Fisher's exact test.

In experiment 1, body weights in group 3 treated with BHP and 2% KA were significantly lower than those in group 2 treated with BHP alone at weeks 4 and 12 (Table IGo). There was no significant difference in body weight between group 2 and untreated control group values. Absolute and relative weights of the thyroid, and relative liver weights in group 3 were significantly increased as compared with group 2 values at weeks 4 and 12. The thyroid weights at week 12 were greater than at week 4. Values for group 2 were similar to those for the untreated control group. There were no significant intergroup differences in the pituitary weights (data not shown). In experiment 2, the thyroid weights were significantly increased in the KA alone group as compared with the control group at weeks 4 and 20.


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Table I. Body, thyroid and liver weights in rats treated with KA after BHP initiation
 
In experiment 1, there were no significant differences in the thyroid-related hormone levels between group 2 and the untreated control group. However, the serum T3 levels in group 3 were significantly lower than those in group 2 at weeks 4 and 12 (Table IIGo). Likewise, the serum T4 levels in group 3 were significantly reduced as compared with group 2 values at weeks 4 and 12. Moreover, the serum TSH levels in group 3 were significantly increased 13–19 times compared with the group 2 values at weeks 4 and 12. The elevation of TSH level in group 3 at week 12 was more remarkable than that at week 4. In experiment 2, similar fluctuations of thyroid-related hormones were observed in the KA alone group at week 4, but not at week 20.


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Table II. Serum thyroid-related hormone levels, liver T4-UDP-GT activities and incidences of thyroid proliferative lesions in rats treated with KA after BHP initiation
 
There were no marked intergroup differences in the liver T4-UDP-GT activities between the untreated control group, group 2 treated with BHP alone and group 3 receiving 2% KA after BHP initiation (Table IIGo).

Incidence data for thyroid proliferative lesions are also given in Table IIGo. No thyroid proliferative lesions were observed in the untreated control group or group 2. In contrast, in group 3, both focal hyperplasias (Figure 2Go) and adenomas (Figure 3Go) were observed at high incidence, the induction generally being significant. In the KA alone group of experiment 2, all animals showed marked diffuse hypertrophy of the thyroid follicular cells at weeks 4 and 20 (Table IIGo).



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Fig. 2. Thyroid follicular hyperplasia in a rat treated with 2% KA for 12 weeks after BHP-initiation, showing increased cellularity of hypertrophic columnar cells. H&E stain, x165.

 


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Fig. 3. Thyroid follicular adenoma in a rat treated with 2% KA for 12 weeks after BHP-initiation, showing expansive growth of follicular cells with basophilic cytoplasm and slightly atypical nuclei. H&E stain, x165.

 
In the present study, continuous KA administration increased thyroid weights and incidences of thyroid proliferative lesions (follicular hyperplasias and adenomas) in rats initiated with BHP. These findings are consistent with the results of the carcinogenicity study in mice performed by Fujimoto et al. (14) and that both rodent species are susceptible to the carcinogenic effects of KA. The observed decrease in serum T3 and T4 levels and marked elevation of serum TSH levels suggest an epigenetic promotion mechanism. Generally, TSH from the pituitary gland plays an important role in enhancement of thyroid tumor development. Its production and release are controlled by thyroid hormone levels in blood via the negative feedback mechanism of the pituitary–thyroid axis (19,20), as proposed by the pioneering work of Bielschowsky (21). Decrease of circulating thyroid hormone levels caused by chronic iodine deficiency or severe thyroid dysfunction leads to an increased release of TSH from the pituitary so that continuous stimulation of the thyroid occurs. In rodents, especially rats, long-term administration of a low iodine diet can cause follicular hyperplasia (20). Iodine deficiency causes reduced production of thyroid hormone, decrease of serum T3 and T4, and increase of serum TSH (22). Moreover, chronic administration of a low iodine diet results in development of thyroid follicular tumors in BHP-initiated rats (23). Many experimental studies have demonstrated similar promoting effects of anti-thyroid substances (2426). For example, propylthiouracil (PTU), an inhibitor of peroxidase-catalyzed iodination (20,27), and thiourea (TU), which blocks iodination of thyrogloblin and the coupling reaction (20) through its action on thyroidal peroxidase, reduce thyroid hormone synthesis. The resultant secretion of TSH promoted development of thyroid follicular proliferative lesions (2426). Hepatic microsomal enzymes may play an important role by catabolizing thyroid hormones so that T4 and T3 levels are lowered and release of TSH is stimulated through the hypothalamic feedback loop (20,28). Phenobarbital (PB), a potent inducer of hepatic microsomal enzymes such as T4-UDP-GT, responsible for T4 clearance in the liver (20,29), is a strong promotor of thyroid carcinogenesis (24,30).

In experiment 1 of our study, the low serum T3 and T4 levels caused by KA administration and increase of serum TSH levels are evidence of the same negative feedback in action. In addition, rats receiving KA without BHP in experiment 2 showed reduction of thyroid hormone levels and elevation of serum TSH level at week 4 (Table IIGo). However, the underlying mechanism remains unclear. Several factors are related to decreased thyroid hormone levels: inhibition of iodine uptake or organic binding of iodide in the thyroid tissue, and enhanced catabolism of thyroid hormones in the liver are possible factors that decrease thyroid hormones (19,20). Increase of hepatic biotransformation by T4-UDP-GT is known to reduce thyroid hormone levels and play a role in thyroid tumor promotion (20,28). But no clear enhancement of liver T4-UDP-GT activity was apparent in rats treated with BHP + KA. These data indicate that this microsomal enzyme found predominantly in the liver and generally recognized to be the major enzyme of glucuronidation is not responsible for the reduction of thyroid hormone levels in rats receiving KA. Therefore, inhibition of iodine uptake or organic iodination in the thyroid by KA may be speculated as a possible mechanism for the reduction of thyroid hormone levels. Additional experiments to clarify this point are now in progress.

KA induced sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells (13), and can cause DNA breaks and clastogenic effect in cultured rat liver cells (8,9), in addition to the mutations of S.typhimurium (1012). Both positive (4) and negative (31) results have been gained for the Rec-assay with Bacillus subtilis. The DNA-repair test in Drosophila and the micronucleus test in mice proved negative (32,33). Although no induction of dominant lethals was observed in an in vivo genotoxicity test, a significant difference from the controls in the numbers of post-implantation losses (early death) was observed in rats receiving KA. It is generally accepted that a reduction of implantation in dominant lethal tests indicate the potency of clastogenic effects on premeiotic stages. Based on these results, KA possesses genotoxic potential in vitro (1012) but there are only few data regarding in vivo effects (11). Although it is still unknown whether the development of thyroid tumors may be influenced by KA genotoxicity, the present study strongly suggests that KA induces thyroid proliferative lesions by a non-genotoxic pathway.


    Notes
 
3 To whom correspondence should be addressed Email: mitsumor{at}nihs.go.jp Back


    Acknowledgments
 
This work was supported in part by a Grant-in Aid for Safety Assessment of Food Additives from the Ministry of Health and Welfare of Japan.


    References
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Received February 17, 1998; revised September 8, 1998; accepted September 25, 1998.