Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan
1 To whom correspondence should be addressed at Division of Pathology, National Institute of Health Sciences, 1181 Kamiyoga, Setagaya-Ku, Tokyo 158-8501, Japan. Fax: +81-337001425. E-mail: imai{at}nihs.go.jp.
Received February 4, 2005; accepted April 8, 2005
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ABSTRACT |
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Key Words: para-aminobenzoic acid; N-bis(2-hydroxypropyl)nitrosamine; thyroid; carcinogenesis; rat.
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INTRODUCTION |
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Para-aminobenzoic acid (PABA) is a component of the water-soluble vitamin B complex, which is widely present in natural foods such as milk, eggs, brown rice, and liver, but its daily requirement for humans is almost all supplied by intestinal bacteria (Altendorf et al., 1969; Dardenne et al., 1975
). PABA is a precursor of folate synthesis, which is catalyzed by dihydropteroate synthase followed by glutamate coupling (Quinlivan et al., 2003
). Sulfonamides, such as sulfamethoxazole, sulfamethazine, sulfamonomethoxine, and sulfadimethoxine (SDM) are analogs of PABA, which structurally resembles para-aminobenzenesulfonamide (sulfamine), the basic skeleton of sulfonamides (Fig. 1). Beneficial effects of sulfonamides are due to their antifolate actions, and they are widely used as antibacterial agents in medical and veterinary practice, whereas their side effect, TPO-inhibition, has also been reported (Doerge and Decker 1994
). On the other hand, it has been known for more than four decades that the structurally similar PABA can show antithyroidal effects (Mccarthy and Murphree, 1960
). Although, details of sulfonamide influence on thyroid carcinogenesis have been well documented, whether PABA also acts as a promoter has remained unclear.
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MATERIALS AND METHODS |
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Animals and experimental design.
Totals of 80 and 21 male F344 rats 6 weeks of age were purchased from Charles River Japan (Kanagawa, Japan) for experiments 1 and 2, respectively, and maintained in a room with a barrier system under the following conditions: temperature 24 ± 1°C, relative humidity 55 ± 5%, ventilation frequency of 18 times/h and a 12 h light/dark cycle. The animals were housed up to five rats per plastic cage, on sterilized soft wood chips (Sankyo Laboratory Service; Tokyo, Japan), and had free access to tap water and powdered basal diet CRF-1 (Oriental Yeast; Tokyo, Japan). After a 1-week acclimatization period, 15 rats each in groups 14 and 10 rats in groups 5 and 6 were allocated with body weight randomization in experiment 1. Rats in groups 14 received a single subcutaneous injection of DHPN at 2800 mg/kg/body weight, and groups 5 and 6 each were given the saline vehicle alone. From 1 week after DHPN initiation, the rats in groups 2, 3, 4, and 6 were fed basal diet containing 0.25%, 0.5%, 1.0%, and 1.0% PABA, respectively, for 40 weeks. Group 1 and 5 animals received basal diet alone (Fig. 2).
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In experiment 2, seven animals each in groups 13 were maintained untreated until 8-weeks of age, and then were given basal diet containing 0.5% and 1.0% PABA diet in groups 2 and 3, respectively, and basal diet alone in group 1, for 2 weeks. General conditions, body weights, and food consumption were recorded as in experiment 1. At the end of the experiment, blood samples were collected from the abdominal aorta of all groups of animals under ether anesthesia for assay of serum T4, T3, and TSH levels with radioimmunoassay kits, T3 RIABEAD Kit for human (Dinabot, North Chicago, IL), GammaCoat Total T4 for human (DiaSorin, Saluggia, Italy), and Rat Thyroid Stimulating Hormone [125I] Biotrak Assay (Amersham Pharmacia Biotech, England, UK), respectively, at SRL (Tokyo, Japan). After euthanasia by exsanguination, thyroids were excised, weighed, and routinely processed for paraffin embedding and preparation of sections stained with H&E. For measurement of proliferative activity of follicular cells, immunohistochemistry for Ki-67 was performed. Briefly, antigen retrieval was achieved by heating in an autoclave for 15 min in citrate buffer at pH 6.0, before exposure to a mouse monoclonal antibody for Ki-67 (clone MIB-5, DAKO), diluted at 1:50, biotinylated rabbit anti mouse-IgG antibody (DAKO), and StreptABComplex/HRP (DAKO). Ki-67-positivity per 1000 follicular cells was assessed to give percentage values.
Statistics.
Variance in data for body weights, organ weights (both absolute and relative weights), and serum hormone levels was checked for homogeneity by Bartlett's procedure. When the data were homogeneous, one-way analysis of variance (ANOVA) was performed. In the heterogeneous cases, the Kruskal-Wallis test was applied. When statistically significant differences were indicated, Dunnett's multiple test was employed for comparison between control and treated groups. For group 5 and 6 data in experiment 1, intergroup differences were analyzed with the Student's or Welch's t-tests. The data for incidences of histopathological findings were analyzed using the Fisher's exact probability test.
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RESULTS |
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Histopathology.
In the thyroids, preneoplastic and neoplastic lesions, including focal follicular hyperplasias, adenomas, and adenocarcinomas (Fig. 3), were dose-dependently increased in groups 24. Incidences of adenomas and adenocarcinomas in groups 3 and 4 were significantly (p < 0.05, 0.01) increased as compared to control group 1 (Table 2). Diffuse follicular cell hyperplasia was not detected in any rats of groups 16. As a proliferative lesion in the liver, altered cell foci were observed on the H&E stain basis (Fig. 4A) in all groups, and their incidences in groups 3 and 4 were significantly (p < 0.05) lower than in the control group 1. However, numbers (group 1: 1.2 ± 1.0; group 4: 2.1 ± 1.5 /cm2) and areas (group 1: 0.07 ± 0.05; group 4: 0.11 ± 0.09 mm2/cm2) of GST-P-positive foci (Fig. 4B) did not vary significantly among the groups. Nephroblastoma in the kidney were apparent in groups 14, and the incidence was slightly increased in group 4. Alveolar/bronchiolar adenomas and adenocarcinomas were frequently observed in the lungs, and atypical tubules, renal cell tumors, or transitional cell tumors in the kidney were apparent in groups 14, but the incidences were not influenced by PABA administration (Table 2). No histopathological changes in the pituitary or nonproliferative lesions in other organs were noted in any of the groups.
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Histopathology.
Diffuse follicular cell hyperplasia was noted in thyroids of five of seven rats in group 2 and all rats of group 3. No focal preneoplastic or neoplastic lesions were detected. Ki-67 positivity: PABA-treated rats in groups 2 and 3 showed significant (p < 0.05, 0.01) increase in Ki-67 positivity as compared to control group 1 (Figs. 5 and 6).
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DISCUSSION |
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Sulfonamides are well known to inhibit the dihydropteroate synthase step of folate synthesis in bacterial cells. This inhibition appears to involve competition between sulfonamides and their structural analog PABA for the same binding site on the enzyme in the presence of adenosine triphosphate and Mg2+ (Brown, 1962; Eagon and McManus, 1989
; Wise and Abou-Dania, 1975
). Although the mechanisms of goitrogenic action of PABA are not completely understood, it has been speculated that TPO inhibition also occurs, given the similarity in chemical structure with sulfamine, both being para-substituted anilines. Sulfamethazine, sulfamine, and other para-substituted anilines reversibly inhibit iodination reactions in vitro catalyzed by lactoperoxidase (LPO), which is closely related to TPO (Doerge and Decker, 1994
). From the available information, the mechanism of goitrogenic action of PABA in the present study is suspected to be reversible alternate substrate inhibition of TPO-catalyzed reactions. However, in another in vivo study using myeloperoxidase, PABA was unexpectedly demonstrated to act as a peroxidase substrate (Kettle and Winterbourn, 1991
). Therefore, it should be clarified whether this might play a role.
The antithyroid action of PABA in the present study can be considered mild in comparison with that of sulfonamides, thioura (TU), or thionamides on the basis of reported changes in serum TSH. For example, SDM treatment for 1 week caused a tenfold or greater elevation, as compared to the no-treatment control (Mitsumori et al., 1995; Onodera et al., 1994
), sulfamonomethoxine for two weeks led to a fivefold increase (Takayama et al., 1986
); TU for 1 week, a tenfold increase (Okuno et al., 1996
; Onodera et al., 1994
; Shimo et al., 1994
); and PTU for 2 or 4 weeks, a tenfold or greater increase (Kitahori et al., 1984
; Mellert et al., 2003
; Takayama et al., 1986
). Because the inhibition constant (Ki value) for PABA regarding peroxidase-catalyzed reactions is much lower than for sulfonamide sulfamethazine (Doerge and Decker, 1994
), the low goitrogenic activity shown in the present study may be due to bioavailability. Regarding blood levels of PABA, we have no data on the dietary administration at doses of 0.25%, 0.5%, and 1.0% in the present study. In the previous literatures, absorption and metabolism of PABA in rats are extremely fast. In the case of an intraduodenal administration at dose of 50 mg/kg to female Wistar rats, time of peak plasma concentration and half-life of elimination phase were 3 min and 114 min, respectively (Staud et al., 1998
). In experiments 1 and 2 of the present study, thyroid weights, incidences of thyroid follicular lesions, and /or serum hormone levels were changed in a dose-dependent manner; hence blood levels of PABA were considered to have increased dose-dependently at dose range of 0.251.0%. Although we should stress that the dose of PABA administered was very high, the action of a primary para-substituted aniline in vivo is of much interest. Further studies are needed to clarify antithyroid effects of other para-substituted anilines, particularly those with electron-donating substituents, which show a high degree of correlation with LPO inhibition (Doerge and Decker, 1994
).
In an experiment with continuous treatment of SDM to rats, serum TSH levels showed rapid and dramatic elevation, which persisted for around 8 weeks from the start of the test but gradually returned to normal thereafter; thyroid follicular cell tumors, including malignant ones, were found to be increased after week 12, with a slight reduction after SDM withdrawal for 4 weeks (Mitsumori et al., 1995). A similar phenomenon regarding serum TSH elevation was also demonstrated in rats treated with TU for 1 or 20 weeks (Onodera et al., 1994
). In the present PABA case, 2-week administration also induced serum TSH elevation, cell proliferative stimulation, and diffuse hyperplasia of thyroid follicular cells. In contrast, diffuse follicular cell hyperplasia in non-preneoplastic/neoplastic parenchyma was not detected at the end of the administration period of 40 weeks, probably indicating return to normal hormone levels.
Carcinogenic target organs of DHPN other than the thyroid include at least the kidney, lung, and liver (Moore et al., 1986). Altered hepatocellular foci, recognized as a preneoplastic lesion, were observed in the liver in all groups, and their incidence in the 0.5% and 1.0% PABA-treated groups significantly decreased as compared to control, on the H&E staining basis, although quantitative analysis of the numbers and areas of GST-P-positive hepatocellular foci revealed no significant intergroup variation. Absolute and relative kidney weights in 1.0% PABA-treated group were significantly increased as compared to control, and these increases are suspected to be a result of increased incidence of nephroblastomas. Similarly, no consistent effects were noted with regard to lung lesions.
The daily requirement of PABA for humans is almost all supplied by intestinal bacteria (Altendorf et al., 1969; Dardenne et al., 1975
), and high consumption of PABA is not recommended. The exposure levels in the present experiments were
215.7 mg/kg/day at 0.5%, in which thyroid tumors were significantly increased, the difference from human exposure being much greater. Thus the risk of toxicity, including thyroid carcinogenesis promotor action for humans, may be negligible.
In conclusion, a stimulatory effect of naturally occurring PABA on rat thyroid carcinogenesis was demonstrated using a two-stage carcinogenesis model with DHPN-initiation. The underlying mechanisms appeared to be associated to some degree with TPO inhibition, resulting in serum TSH stimulation, a finding in line with the chemical structural similarity with sulfonamides.
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ACKNOWLEDGMENTS |
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