* School of Pharmaceutical Sciences and COE Program in the 21st Century, University of Shizuoka, 52-1, Yada, Shizuoka 422-8526, Japan; Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan;
Daiichi College of Pharmaceutical Sciences, 22-1, Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan;
Center for Biological Safety & Research, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
Received May 17, 2004; accepted June 22, 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: polychlorinated biphenyls; Kanechlor-500; thyroid hormones; UDP-glucuronosyltransferases; Wistar rats; Gunn rats.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, therefore, we examined a relationship between the decrease in serum total T4 level and the increase in the hepatic T4-UDP-GT (UGT1A1 and UGT1A6) by PCB using Wistar and UGT1A-deficient Wistar rats (Gunn rats). In this way, we demonstrated that the PCB-mediated decrease in serum total T4 level in rats was not necessarily dependent on the increase in hepatic T4-glucuronidation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal treatments. Male Wistar rats (160200 g) and UGT1A-deficient Wistar rats (Gunn rats, 190260 g) were obtained from Japan SLC., Inc. (Shizuoka, Japan). Male Wistar and Gunn rats were housed three or four per cage with free access to commercial chow and tap water, and were maintained on a 12-h dark/light cycle (8:00 a.m.8:00 p.m. light) in an air-controlled room (temperature: 24.5 ± 1°C , humidity: 55 ± 5%), and were handled with humane care under the guidelines of the University of Shizuoka (Shizuoka, Japan). Treatments of rats with KC500 (100 mg/kg) and PentaCB (112 mg/kg) were performed according to the method of Kato et al. (2001, 2003
). Briefly, the rats received a single ip injection of KC500 (100 mg/kg) or PentaCB (112 mg/kg) dissolved in Panacete 810 (5 ml/kg). Control animals were treated with vehicle alone (5 mg/kg).
Analysis of serum hormones. All rats were killed by decapitation on day 4 after the dosing, and the liver was removed. Blood was collected from each animal between 10:30 and 11:30 a.m. After clotting at room temperature, serum was separated by centrifugation and stored at 50°C until used. The levels of total T4, total triiodothyronine (T3), free T4 and thyroid-stimulating hormone (TSH) were measured by radioimmunoassay using the T-4· and T-3· RIABEAD (DAINABOT Co., Ltd, Tokyo, Japan), free T4 (Diagnostic Products Corporation; Los Angels, CA), and Biotrak rTSH [125I] assay system (Amersham Life Science Ltd.; Little Chalfont, UK), respectively.
Hepatic microsomal UDP-GT and deiodinase assays. Hepatic microsomes were prepared according to the method of Kato et al. (1995). The amount of protein was determined by the method of Lowry et al. (1951)
with bovine serum albumin as standard. The activities of microsomal UDP-GT toward T4 and chloramphenicol were determined by the methods of Barter and Klaassen (1992)
and Ishii et al. (1994)
, respectively. All UDP-GT activities were measured after activation of the UDP-GTs by 0.05% Brij 58. The activity of hepatic microsomal type I outer-ring deiodinase was determined by the method of Hood and Klaassen (2000)
.
Western blot analysis. Polyclonal anti-peptide antibodies against the common region of UGT1A isoforms and a specific antibody against UGT1A1, UGT1A6, or UGT2B1 were used (Ikushiro et al. 1995, 1997
). Western analyses for microsomal UGT isoforms were performed by the method of Luquita et al. (2001)
. The detection of protein was performed using a chemical luminescence (ECL detection kit, Amersham Pharmacia Biotech), and the band intensity was quantified densitometrically with LAS-1000 (FUJIFILM, Japan).
Determination of hydroxylated PCB metabolites in the serum. The extraction and sample clean-up procedures for serum PCB metabolites were preformed by the method of Haraguchi et al. (1998). The identification of hydroxylated PCB metabolites was carried out on a GC/MS system (GC-17A, QP-5000, Shimadzu, Japan) with a DB-5 capillary column (60 m x 0.25 mm, i.d.). The temperature program was as follows: 100°C, 2 min, 100250°C at 20°C/min, 250280°C at 2°C/min (Mimura et al., 1999
). Quantification of the hydroxylated PCB metabolites was performed on GC/ECD (GC-14A, Shimadzu, Japan) by comparison with an internal standard of 2,2',3,4',5,5',6-heptachloro-4-[13C]biphenylol. The major hydroxylated PCB metabolites (>5 ng/g liver) were analyzed.
Statistics. The data obtained were statistically analyzed according to Dunnett's test after the analysis of variance (ANOVA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In general, PCBs, including 3,3',4,4',5-pentachlorobiphenyl, 2,2',4,4',5,5'-hexachlorobiphenyl, and Aroclor 1254 have been thought to decrease the level of serum T4 through increase in the activity of hepatic T4-UDP-GT (Barter and Klaassen, 1994; Schuur et al., 1997
; Van Birgelen et al., 1995
). However, it has been reported that the difference between rats and mice in magnitude of decrease in level of serum total T4 by 2,2',4,4',5,5'-hexachlorobiphenyl is not well correlated with that of increase in activity of T4-UDP-GT (Craft et al., 2002
). Furthermore, we have found that KC500 resulted in a significant decrease in the serum T4 level in both rats and mice, although a significant increase in T4-UDP-GT activity occurred only in rats but not in mice (Kato et al., 2003
). In addition, a decrease in the serum level of total T4 by PentaCB or 2,2',3,3',4,6'-hexachlorobiphenyl occurred in both rats and mice, although a significant change in activity of UDP-GT, specially UGT1A6, was hardly observed in the both species (Kato et al., 2001
). These previous reports strongly support the finding that the decrease in serum total T4 level by PCB does not occur only through an increase in hepatic T4-UDP-GT activity.
As possible mechanisms for the PCB-mediated decrease in serum T4, changes in type-I deiodinase activity and serum TSH level might also be considered. However, no increase (significant decrease) in hepatic activity of microsomal type-I deiodinase, which mediates the deiodization of T4 and T3, was observed in either Wistar or Gunn rats. Similar results have been reported in previous study using Aroclor 1254-treated Sprague-Dawley rats (Hood and Klassen, 2000). Accordingly, a PCB-mediated decrease in serum T4 level is thought to occur through a type-I deiodinase-independent pathway. Furthermore, the level of serum TSH in both Wistar and Gunn rats was not significantly changed by either KC500 or PentaCB, indicating that TSH is not related to the PCB-mediated decrease in the serum T4 level. In addition, it had been reported that the serum TSH level was little affected by PCB (Hallgren et al., 2001
; Hood et al., 1999
; Liu et al., 1995
; Kato et al., 2003
).
As another possible mechanism, binding of hydroxylated PCBs to TTR, a major T4-transporting protein, might be considered, (1) because hydroxylated PCB metabolites show the binding affinity for TTR (Brouwer et al., 1998; Lans et al., 1993
) and (2) because the binding affinity of 4-OH-2,3,3',4',5-pentachlorobiphenyl, which was detected as a main hydroxylated metabolite in KC500-treated rats in the present experiments, is 3.3-fold higher than that of the natural ligand T4 (Meerts et al., 2002
). The present findings and previous reports suggest that the decrease in the level of serum T4 in either KC500-treated or PentaCB-treated Wistar and Gunn rats might occur, at least in part, through a TTR-associated pathway. Furthermore, dihydroxylated PCBs have been reported to show a several fold higher affinity for TTR than monohydroxylated PCBs (Lans et al., 1993
). In KC500-treated Gunn rats, the sum of three dihydroxylated PCB metabolites was 37% of the total hydroxylated PCB metabolites detected, although in the PCB-treated Wistar rats, the dihydroxylated metabolite was hardly detected. In addition, in PentaCB-treated Wistar and Gunn rats, the amount of 3',4'-(OH)2-PentaCB was more than 80% of the total hydroxylated PCB metabolites detected in the serum. Furthermore, PentaCB, which shows a weaker affinity for TTR than natural T4 (Chauhan et al., 2000
), was also detected in the serum at a low level, as compared with the total hydroxylated metabolites. Accordingly, the binding of dihydroxylated PCB metabolites and PentaCB to TTR might also be attributed, in part, to a decrease in the level of serum T4 in either KC500-treated or PentaCB-treated rats. However, an increase in the serum free T4 level did not occur in any rats treated with either KC500 or PentaCB, although Pedraza and colleagues (1996)
have shown that the synthetic flavinoid EM-21388, which displaces T4 from TTR, increases the serum free T4 level. Considering the hydroxylated metabolites of the PCBs examined, the decrease in serum total T4 level by KC500 or PentaCB seems to occur, at least in part, through a TTR-associated pathway, although the reason that the serum level of free T4 was decreased remains unclear. Furthermore, two other factors might be considered as possible mechanisms for the PCB-mediated decrease in the level of serum T4: (1) the change in the performance of the hypothalamo-pituitary-thyroid-axis (Khan et al., 2002
; Khan and Hansen, 2003
) and (2) the increase in estrogen sulfotransferase, which efficiently catalyzes the sulfation of iodothyronines (Kester et al., 1999
). However, the exact mechanisms for the PCB-mediated decrease in the serum T4 level remains unclear.
In conclusion, the present findings demonstrate that the decrease in serum total T4 level by PCB in Gunn rats occurs without an increase in hepatic T4-UDP-GT activity; they further suggest that in rats, especially Gunn rats, the PCB-mediated decrease might occur, at least in part, through formation of the hydroxylated PCB metabolites. In Wistar rats, however, the PCB-mediated induction of T4-UDP-GT might also contribute to the decrease. Further studies are necessary for understanding the susceptibility toward a PCB-mediated decrease in serum T4 level in animals, including humans.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed at School of Pharmaceutical Sciences and COE Program in the 21st Century, University of Shizuoka, 52-1, Yada, Shizuoka 422-8526, Japan. Fax: +81 54 264 56 35. E-mail: kato{at}ys7.u-shizuoka-ken.ac.jp
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barter, R. A., and Klaassen, C. D. (1994). Reduction of thyroid hormone levels and alteration of thyroid function by four representative UDP-glucuronosyltransferase inducers in rats. Toxicol. Appl. Pharmacol. 128, 917.[CrossRef][ISI][Medline]
Brouwer, A., Morse, D. C., Lans, M. C., Schuur, A. G., Murk, A. J., Klasson-Wehler, E., Bergman, Å., and Visser, T. J. (1998). Interactions of persistent environmental organohalogens with the thyroid hormone system: Mechanisms and possible consequences for animal and human health. Toxicol. Ind. Health 14, 5984.[ISI][Medline]
Cadogan, J. I. G. (1962). A convenient new method of aromatic arylation. J. Chem. Soc. 42574258.
Chauhan, K. R., Kodavanti, P. R. S., and McKinney, J. D. (2000). Assessing the role of ortho-substitution on polychlorinated biphenyl binding to transthyretin, a thyroxine transport protein. Toxicol. Appl. Pharmacol. 162, 1021.[CrossRef][ISI][Medline]
Collins, W. T. Jr., and Capen, C. C. (1980). Biliary excretion of 125I-thyroxine and fine structural alterations in the thyroid glands of Gunn rats fed polychlorinated biphenyls (PCB). Lab. Invest. 43, 158164.[ISI][Medline]
Craft, E. S., DeVito, M. J., and Crofton, K. M. (2002). Comparative responsiveness of hypothyroxinemia and hepatic enzyme induction in Long-Evans rats versus C57BL/6 J mice exposed to TCDD-like and phenobarbital-like polychlorinated biphenyl congeners. Toxicol. Sci. 68, 372380.
De Sandro, V., Catinot, R., Kriszt, W., Cordier, A., and Richert, L. (1992). Male rat hepatic UDP-glucuronosyltransferase activity toward thyroxine. Activation and induction properties-Relation with thyroxine plasma disappearance rate. Biochem. Pharmacol. 43, 15631569.[CrossRef][ISI][Medline]
Hallgren, S., Sinjari, T., Håkansson, H., and Darnerud, P. O. (2001). Effects of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) on thyroid hormone and vitamin A levels in rats and mice. Arch. Toxicol. 75, 200208.[CrossRef][ISI][Medline]
Haraguchi, K., Kato, Y., Kimura, R., and Masuda, Y. (1998). Hydroxylation and methylthiolation of mono-ortho-substituted polychlorinated biphenyls in rats: Identification of metabolites with tissue affinity. Chem. Res. Toxicol. 11, 15081515.[CrossRef][ISI][Medline]
Hood, A., Allen, M. L., Liu, Y., Liu, J., and Klaassen, C. D. (2003). Induction of T4 UDP-GT activity, serum thyroid stimulating hormone, and thyroid follicular cell proliferation in mice treated with microsomal enzyme inducers. Toxicol. Appl. Pharmacol. 188, 613.[CrossRef][ISI][Medline]
Hood, A., Hashmi, R., and Klaassen, C. D. (1999). Effects of microsomal enzyme inducers on thyroid-follicular cell proliferation, hyperplasia, and hypertrophy. Toxicol. Appl. Pharmacol. 160, 163170.[CrossRef][ISI][Medline]
Hood, A., and Klaassen, C. D. (2000). Effects of microsomal enzyme inducers on outer-ring deiodinase activity toward thyroid hormones in various rat tissues. Toxicol. Appl. Pharmacol. 163, 240248.[CrossRef][ISI][Medline]
Ikushiro, S., Emi, Y., and Iyanagi, T. (1995). Identification and analysis of drug-responsive expression of UDP-glucuronosyltransferase family 1 (UGT1) isozyme in rat hepatic microsomes using anti-peptide antibodies. Arch. Biochem. Biophys. 324, 267272.[CrossRef][ISI][Medline]
Ikushiro, S., Emi, Y., and Iyanagi, T. (1997). Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes. Biochemistry 36, 71547161.[CrossRef][ISI][Medline]
Ishii, Y., Tsuruda, K., Tanaka, M., and Oguri, K. (1994). Purification of a phenobarbital-inducible morphine UDP-glucuronyltransferase isoform, absent from Gunn rat liver. Arch. Biochem. Biophys. 315, 345351.[CrossRef][ISI][Medline]
Kato, Y., Haraguchi, K., Kawashima, M., Yamada, S., Masuda, Y., and Kimura, R. (1995). Induction of hepatic microsomal drug metabolizing enzymes by methylsulphonyl metabolites of polychlorinated biphenyl congeners in rats. Chem.-Biol. Interact. 95, 257268.[CrossRef][ISI][Medline]
Kato, Y., Haraguchi, K., Yamazaki, T., Ito, Y., Miyajima, S., Nemoto, K., Koga, N., Kimura, R., and Degawa, M. (2003). Effects of polychlorinated biphenyls, Kanechlor-500, on serum thyroid hormone levels in rats and mice. Toxicol. Sci. 72, 235241.
Kato, Y., Yamazaki, T., Haraguchi, K., Ito, Y., Nemoto, K., Masuda, Y., Degawa, M., and Kimura, R. (2001). Effects of 2,2',4,5,5'-pentachlorobiphenyl and 2,2',3,3',4,6'-hexachlorobiphenyl on serum hormone levels in rats and mice. Organohalogen Compounds 53, 4446.
Kester, M. H. A., van Dijk, C. H., Tibboel, D., Hood, A. M., Rose, N. J. M., Meinl, W., Pabel, U., Glatt, H., Falany, C. N., Coughtrie, M. W. H. et al. (1999). Sulfation of thyroid hormone by estrogen sulfotransferase. J. Clin. Endocrinol. Metab. 84, 25772580.
Khan, M. A., and Hansen, L. G. (2003). ortho-Substituted polychlorinated biphenyl (PCB) congeners (95 or 101) decrease pituitary response to thyrotropin releasing hormone. Toxicol. Lett. 144, 173182.[ISI][Medline]
Khan, M. A., Lichtensteiger, C. A., Faroon, O., Mumtaz, M., Schaeffer, D. J., and Hansen, L. G. (2002). The hypothalamo-pituitary-thyroid (HPT) axis: A target of nonpersistent ortho-substituted PCB congeners. Toxicol. Sci. 65, 5261.
Lans, M. C., Klasson-Wehler, E., Willemsen, M., Meussen, E., Safe, S., and Brouwer, A. (1993). Structure-dependent, competitive interaction of hydroxy-polychlorobiphenyls, -dibenzo-p-dioxins and -dibenzofurans with human transthyretin. Chem.-Biol. Interact. 88, 721.[CrossRef][ISI][Medline]
Li, M.-H., Hsu, P.-C., and Guo, Y. L. (2001). Hepatic enzyme induction and acute endocrine effects of 2,2',3,3',4,6'-hexachlorobiphenyl and 2,2',3,4',5',6-hexachlorobiphenyl in prepubertal female rats. Arch. Environ. Contam. Toxicol. 41, 381385.[CrossRef][ISI][Medline]
Liu, J., Liu, Y., Barter, R. A., and Klaassen, C. D. (1995). Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: A dose-response study. J. Pharmacol. Exp. Ther. 273, 977985.[Abstract]
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
Luquita, M. G., Catania, V. A., Pozzi, E. J. S., Veggi, L. M., Hoffman, T., Pellegrino, J. M., Ikushiro, S., Emi, Y., Iyanagi, T., Vore, M. et al. (2001). Molecular basis of perinatal changes in UDP-glucuronosyltransferase activity in maternal rat liver. J. Pharmacol. Exp. Ther. 298, 4956.
Mimura, K., Tamura, M., Haraguchi, K., and Masuda, Y. (1999). Analysis of 209 PCB congeners by high separation gas chromatography/low resolution mass spectrometer. Fukuoka Acta Med. 90, 192201.[Medline]
Meerts, I. A. T. M., Assink, Y., Cenijn, P. H., van den Berg, J. H. J., Weijers, B. M., Bergman, Å., Koeman, J. H., and Brouwer, A. (2002). Placental transfer of a hydroxylated polychlorinated biphenyl and effects on fetal and maternal thyroid hormone homeostasis. Toxicol. Sci. 68, 361371.
Ness, D. K., Schantz, S. L., Moshtaghian, J., and Hansen, L. G. (1993). Effects of perinatal exposure to specific PCB congeners on thyroid hormone concentrations and thyroid histology in the rat. Toxicol. Lett. 68, 311323.[CrossRef][ISI][Medline]
Pedraza, P., Calvo, R., Obregón, M. J., Asunción, M., Escobar del Rey, F., and Morreale de Escobar, G. (1996). Displacement of T4 from transthyretin by the synthetic flavonoid EMD 21388 results in increased production of T3 from T4 in rat dams and fetuses. Endocrinology 137, 49024914.[Abstract]
Saito, K., Kaneko, H., Sato, K., Yoshitake, A., and Yamada, H. (1991). Hepatic UDP-glucuronyltransferase(s) activity toward thyroid hormones in rats: Induction and effects on serum thyroid hormone levels following treatment with various enzyme inducers. Toxicol. Appl. Pharmacol. 111, 99106.[ISI][Medline]
Schuur, A. G., Boekhorst, F. M., Brouwer, A., and Visser, T. J. (1997). Extrathyroidal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on thyroid hormone turnover in male Sprague-Dawley rats. Endocrinology 138, 37273734.
van Birgelen, A. P. J. M., Smit, E. A., Kampen, I. M., Groeneveld, C. N., Fase, K. M., van der Kolk, J., Poiger, H., van den Berg, M., Koeman, J. H., and Brouwer, A. (1995). Subchronic effects of 2,3,7,8-TCDD or PCBs on thyroid hormone metabolism: Use in risk assessment. Eur. J. Pharmacol. 293, 7785.[Medline]
Visser, T. J. (1996). Pathways of thyroid hormone metabolism. Acta Med. Austriaca 1/2, 1016.
Visser, T. J., Kaptein, E., van Toor, H., van Raaij, J. A. G. M., van den Berg, K. J., Joe, C. T. T., van Engelen, J. G. M., and Brouwer, A. (1993). Glucuronidation of thyroid hormone in rat liver: Effects of in vivo treatment with microsomal enzyme inducers and in vitro assay conditions. Endocrinology 133, 21772186.[Abstract]