Promotion of Thyroid Carcinogenesis by para-aminobenzoic Acid in Rats Initiated with N-bis(2-hydroxypropyl)nitrosamine
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《毒物学科学杂志》
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan
ABSTRACT
Sulfonamide analogues of para-aminobenzoic acid (PABA), a precursor of folate synthesis, have beneficial effects as antifolate, but thyroid peroxidase inhibition has been reported as a side effect that results in promotion of rat thyroid carcinogenesis. In the present study, effects of PABA itself on F344 rat thyroid carcinogenesis after initiation with N-bis(2-hydroxypropyl)nitrosamine (DHPN) were evaluated. In experiment 1, rats in groups 1–4
Key Words: para-aminobenzoic acid; N-bis(2-hydroxypropyl)nitrosamine; thyroid; carcinogenesis; rat.
INTRODUCTION
Certain classes of chemicals have been demonstrated to inhibit thyroid peroxidase (TPO), which catalyzes iodination of tyrosyl residues in thyroglobulin and coupling reactions of iodotyrosines in thyroid hormone biosynthesis (Capen, 2001). Sulfonamide, aniline derivatives are included, these inducing goiter and thyroid tumors via interference with thyroid hormone synthesis followed by increase in the serum thyroid stimulating hormone (TSH) level in rats (Littlefield et al., 1990; Swarm et al., 1973; Takayama et al., 1986).
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.
In the present study, modifying effects of PABA on thyroid carcinogenesis were evaluated in a 40-week two-stage rat model using N-bis(2-hydroxypropyl)nitrosamine-initiation (DHPN), which has been extensively applied to detect modifying effects of various compounds within a relatively short period (Hiasa et al., 1982). We earlier reported SDM to induce thyroid follicular cell tumors, including invasive adenocarcinomas after DHPN-initiation (Imai et al., 2005; Mitsumori et al., 1995). To cast light on mechanisms of action, thyroid weight, serum thyroxine (T4), triiodothyronine (T3), and TSH levels and proliferative activity of thyroid follicular cells in rats treated with PABA were also measured after a short period of exposure.
MATERIALS AND METHODS
Chemicals.
PABA (purity > 99.5%) and DHPN were purchased from Sigma Chemical (St. Louis, MO) and Nacalai Tesque (Kyoto, Japan), respectively.
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 1–4 and 10 rats in groups 5 and 6 were allocated with body weight randomization in experiment 1. Rats in groups 1–4
General conditions were noted daily, and body weights and food consumption were measured weekly during the experiment. At the end of the experiment period, all animals were euthanized by exsanguination from the abdominal aorta under ether anesthesia. The thyroids, pituitary, liver, kidneys, and lungs of each animal were excised and weighed. As for the thyroids and kidneys, weights of each side were separately recorded and the total of the two sides was used for calculation of group mean and SD values. Paraffin-embedded sections were routinely prepared and stained with hematoxylin and eosin (H&E) for histopathological observation. Based on the histopathological findings for livers in PABA-treated groups of the present study, immunohistochemistry for glutathione S-transferase placental form (GST-P; Ito et al., 1988; Takizawa et al., 2004) to the liver paraffin sections in groups 1 and 4 was also performed. Briefly, rabbit polyclonal antibodies for GST-P (MBL; Nagoya, Japan) were used at a dilution of 1:1000, with biotinylated goat anti-rabbit immunoglobulins (DAKO A/S; Glostrup, Denmark) and StreptABComplex/HRP (DAKO). Reaction products were developed by immersing the sections in 3,3'-diaminobenzidine 4HCl containing hydrogen peroxide. Sections were lightly counterstained with hematoxylin for microscopic examination. Positive reactions on all specimens were confirmed in bile duct epithelia (Imai et al., 1997). Negative controls without primary antibody reactions were set for using serial sections. Numbers and areas of GST-P–positive liver cell foci larger than 0.2 mm in diameter were quantified with the aid of an image analyzer IPAP (Sumika Technos; Osaka, Japan).
In experiment 2, seven animals each in groups 1–3 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.
RESULTS
Experiment 1
Mortality, body weights, and food consumption.
One animal of group 1 died of a renal nephroblastoma at week 26, and thus was excluded from all evaluations. Daily food consumption was similar among all groups, resulting in a good correlation between the targeted dose of PABA and the actual daily intake (Table 1). No body weight change with PABA administration was observed in rats with or without DHPN-initiation (Table 1).
Organ weights.
Absolute and relative thyroid weights were dose-dependently increased, and statistical significance (p < 0.01) was noted in group 4 as compared to control group 1, at least partially reflecting tumor development. Thyroid were also significantly (p < 0.01) increased in group 6 as compared to group 5, but to a lesser extent than in group 4 (Table 1). Absolute kidney weights in group 4 and relative values in groups 3 and 4 were significantly (p < 0.05) increased as compared to control group 1. Statistically significant changes were not evident for the pituitary, liver and lung (Table 1).
Histopathology.
In the thyroids, preneoplastic and neoplastic lesions, including focal follicular hyperplasias, adenomas, and adenocarcinomas (Fig. 3), were dose-dependently increased in groups 2–4. 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 1–6. 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 1–4, 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 1–4, 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.
Experiment 2
Mortality, body weight, and food consumption.
No mortality or body weight change due to PABA administration was observed (Table 3). Daily food consumption was similar among all groups, resulting in a good correlation between the targeted dose of PABA and the actual daily intake (Table 3).
Organ weights.
Absolute and relative thyroid weights in group 3 were significantly (p < 0.01) increased as compared to values in control group 1 (Table 3).
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).
Serum hormone levels:
Although no change in T3 were detected in groups 2 and 3, the T4 level was significantly (p < 0.05) depressed in group 3, and TSH levels were significantly (p < 0.05, 0.01) elevated in a dose-dependent manner in groups 2 and 3, as compared to control group 1 (Table 4).
DISCUSSION
The present two-stage rat thyroid carcinogenesis model with DHPN-initiation has been widely used for detection of thyroid carcinogens, as well as for promoters exerting antithyroidal activities resulting in TSH stimulation via negative feedback through the pituitary–thyroid axis (Mitsumori et al., 1995; Yasuhara et al., 2001). In experiment 1 of the present study, dietary PABA administration significantly increased thyroid weights along with the incidence of follicular cell adenomas and adenocarcinomas, clearly indicating the compound to exert promotion/progression actions on rat thyroid carcinogenesis. To investigate whether the stimulatory effect was due to antithyroid effects, serum T4, T3, and TSH levels were determined in experiment 2. Although the T3 level was not changed, T4 was significantly depressed, and the TSH values were elevated in rats treated with PABA for 2 weeks. In addition, thyroid weights and Ki-67 positivity were significantly increased. Accordingly, a goitrogenic action through increase in TSH level was confirmed to have contributed, at least in part, to the stimulation of thyroid carcinogenesis.
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 0.25–1.0%. 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.
ACKNOWLEDGMENTS
This study was supported by a grant-in-aid from the Ministry of Health, Labour and Welfare of Japan.
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ABSTRACT
Sulfonamide analogues of para-aminobenzoic acid (PABA), a precursor of folate synthesis, have beneficial effects as antifolate, but thyroid peroxidase inhibition has been reported as a side effect that results in promotion of rat thyroid carcinogenesis. In the present study, effects of PABA itself on F344 rat thyroid carcinogenesis after initiation with N-bis(2-hydroxypropyl)nitrosamine (DHPN) were evaluated. In experiment 1, rats in groups 1–4
Key Words: para-aminobenzoic acid; N-bis(2-hydroxypropyl)nitrosamine; thyroid; carcinogenesis; rat.
INTRODUCTION
Certain classes of chemicals have been demonstrated to inhibit thyroid peroxidase (TPO), which catalyzes iodination of tyrosyl residues in thyroglobulin and coupling reactions of iodotyrosines in thyroid hormone biosynthesis (Capen, 2001). Sulfonamide, aniline derivatives are included, these inducing goiter and thyroid tumors via interference with thyroid hormone synthesis followed by increase in the serum thyroid stimulating hormone (TSH) level in rats (Littlefield et al., 1990; Swarm et al., 1973; Takayama et al., 1986).
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.
In the present study, modifying effects of PABA on thyroid carcinogenesis were evaluated in a 40-week two-stage rat model using N-bis(2-hydroxypropyl)nitrosamine-initiation (DHPN), which has been extensively applied to detect modifying effects of various compounds within a relatively short period (Hiasa et al., 1982). We earlier reported SDM to induce thyroid follicular cell tumors, including invasive adenocarcinomas after DHPN-initiation (Imai et al., 2005; Mitsumori et al., 1995). To cast light on mechanisms of action, thyroid weight, serum thyroxine (T4), triiodothyronine (T3), and TSH levels and proliferative activity of thyroid follicular cells in rats treated with PABA were also measured after a short period of exposure.
MATERIALS AND METHODS
Chemicals.
PABA (purity > 99.5%) and DHPN were purchased from Sigma Chemical (St. Louis, MO) and Nacalai Tesque (Kyoto, Japan), respectively.
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 1–4 and 10 rats in groups 5 and 6 were allocated with body weight randomization in experiment 1. Rats in groups 1–4
General conditions were noted daily, and body weights and food consumption were measured weekly during the experiment. At the end of the experiment period, all animals were euthanized by exsanguination from the abdominal aorta under ether anesthesia. The thyroids, pituitary, liver, kidneys, and lungs of each animal were excised and weighed. As for the thyroids and kidneys, weights of each side were separately recorded and the total of the two sides was used for calculation of group mean and SD values. Paraffin-embedded sections were routinely prepared and stained with hematoxylin and eosin (H&E) for histopathological observation. Based on the histopathological findings for livers in PABA-treated groups of the present study, immunohistochemistry for glutathione S-transferase placental form (GST-P; Ito et al., 1988; Takizawa et al., 2004) to the liver paraffin sections in groups 1 and 4 was also performed. Briefly, rabbit polyclonal antibodies for GST-P (MBL; Nagoya, Japan) were used at a dilution of 1:1000, with biotinylated goat anti-rabbit immunoglobulins (DAKO A/S; Glostrup, Denmark) and StreptABComplex/HRP (DAKO). Reaction products were developed by immersing the sections in 3,3'-diaminobenzidine 4HCl containing hydrogen peroxide. Sections were lightly counterstained with hematoxylin for microscopic examination. Positive reactions on all specimens were confirmed in bile duct epithelia (Imai et al., 1997). Negative controls without primary antibody reactions were set for using serial sections. Numbers and areas of GST-P–positive liver cell foci larger than 0.2 mm in diameter were quantified with the aid of an image analyzer IPAP (Sumika Technos; Osaka, Japan).
In experiment 2, seven animals each in groups 1–3 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.
RESULTS
Experiment 1
Mortality, body weights, and food consumption.
One animal of group 1 died of a renal nephroblastoma at week 26, and thus was excluded from all evaluations. Daily food consumption was similar among all groups, resulting in a good correlation between the targeted dose of PABA and the actual daily intake (Table 1). No body weight change with PABA administration was observed in rats with or without DHPN-initiation (Table 1).
Organ weights.
Absolute and relative thyroid weights were dose-dependently increased, and statistical significance (p < 0.01) was noted in group 4 as compared to control group 1, at least partially reflecting tumor development. Thyroid were also significantly (p < 0.01) increased in group 6 as compared to group 5, but to a lesser extent than in group 4 (Table 1). Absolute kidney weights in group 4 and relative values in groups 3 and 4 were significantly (p < 0.05) increased as compared to control group 1. Statistically significant changes were not evident for the pituitary, liver and lung (Table 1).
Histopathology.
In the thyroids, preneoplastic and neoplastic lesions, including focal follicular hyperplasias, adenomas, and adenocarcinomas (Fig. 3), were dose-dependently increased in groups 2–4. 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 1–6. 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 1–4, 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 1–4, 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.
Experiment 2
Mortality, body weight, and food consumption.
No mortality or body weight change due to PABA administration was observed (Table 3). Daily food consumption was similar among all groups, resulting in a good correlation between the targeted dose of PABA and the actual daily intake (Table 3).
Organ weights.
Absolute and relative thyroid weights in group 3 were significantly (p < 0.01) increased as compared to values in control group 1 (Table 3).
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).
Serum hormone levels:
Although no change in T3 were detected in groups 2 and 3, the T4 level was significantly (p < 0.05) depressed in group 3, and TSH levels were significantly (p < 0.05, 0.01) elevated in a dose-dependent manner in groups 2 and 3, as compared to control group 1 (Table 4).
DISCUSSION
The present two-stage rat thyroid carcinogenesis model with DHPN-initiation has been widely used for detection of thyroid carcinogens, as well as for promoters exerting antithyroidal activities resulting in TSH stimulation via negative feedback through the pituitary–thyroid axis (Mitsumori et al., 1995; Yasuhara et al., 2001). In experiment 1 of the present study, dietary PABA administration significantly increased thyroid weights along with the incidence of follicular cell adenomas and adenocarcinomas, clearly indicating the compound to exert promotion/progression actions on rat thyroid carcinogenesis. To investigate whether the stimulatory effect was due to antithyroid effects, serum T4, T3, and TSH levels were determined in experiment 2. Although the T3 level was not changed, T4 was significantly depressed, and the TSH values were elevated in rats treated with PABA for 2 weeks. In addition, thyroid weights and Ki-67 positivity were significantly increased. Accordingly, a goitrogenic action through increase in TSH level was confirmed to have contributed, at least in part, to the stimulation of thyroid carcinogenesis.
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 0.25–1.0%. 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.
ACKNOWLEDGMENTS
This study was supported by a grant-in-aid from the Ministry of Health, Labour and Welfare of Japan.
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