| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
RESEARCH REPORT |
1 Department of Pedodontics and Orthodontics, Institute of Dentistry, University of Helsinki, Finland;
2 Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Finland;
3 Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Finland;
4 Kuopio Department, National Veterinary and Food Research Institute, Finland;
5 Department of Environmental Health, National Public Health Institute, Kuopio, Finland;
6 Department of Oral Pathology, Institute of Dentistry, University of Helsinki, Finland;
7 Department of Pathology, Helsinki University Central Hospital, Finland;
10 corresponding author, Institute of Dentistry, Biomedicum Helsinki, PO Box 63, FIN-00014 UNIVERSITY OF HELSINKI, Finland, carin.sahlberg{at}helsinki.fi
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
KEY WORDS: TCDD • enamel maturation • dentin mineralization • AhR • CYP1A1
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 2000). The detailed mechanisms underlying the spectrum of toxic responses elicited by TCDD are poorly understood. Several studies indicate that the effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR) (Nebert et al., 2000). AhR is a cytosolic protein, which belongs to a family of transcriptional regulatory proteins containing the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) domain structure (Gu et al., 2000). An important feature of AhR, distinguishing it from other members of this protein family, is that around the C-terminal end of its PAS domain, it possesses a ligand-binding subunit for high-affinity binding to TCDD (Schmidt and Bradfield, 1996). Experiments on AhR knockout mice show that, for the most part, TCDD toxicity is not observed in mice lacking a functional AhR gene (Fernandez-Salguero et al., 1996). Versatility of AhR-related biological responses implies that signal transduction of AhR involves a complex process, during which AhR expression is regulated in a tissue-, species-, and developmental-stage-dependent manner.
Cytochrome P450 enzymes play important roles in drug, toxicant, steroid hormone, and fatty acid metabolism. Some substrates like TCDD can induce their own metabolism by regulating cytochrome P450 transcription. Cytochrome P4501A1 (CYP1A1) is a substrate-inducible microsomal enzyme, which oxygenates TCDD and related compounds as the initial step in their metabolic processing to water-soluble derivatives for excretion from the body (Whitlock, 1999). In addition, the induction of CYP1A1 is thought to be a paradigm of gene regulation by TCDD. Upon TCDD binding, AhR translocates into the nucleus, forms a heterodimer with AhR nuclear translocator (ARNT, another bHLH/PAS transcription factor), and induces the transcription of the CYP1A1 gene.
Han/Wistar (Kuopio; H/W) rats are exceptionally resistant to the acute lethality of TCDD, but the strain still exhibits many biochemical and toxic responses to TCDD, such as CYP1A1 induction, thymic atrophy, and teratogenic and developmental effects comparable with those seen in other strains (Huuskonen et al., 1994; Pohjanvirta et al., 1998; Kiukkonen et al., 2002). To guarantee maximal response, we chose H/W rats to test our hypothesis that TCDD interferes with mineralization of the dental matrices.
|
MATERIALS & METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. The use of animals was approved by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Science of the University of Helsinki.
|
Antibodies
Specificity of the polyclonal rabbit antibodies to rat AhR has been established (Pohjanvirta et al., 1998). Polyclonal rabbit antibodies to rat CYP1A1 were from Chemicon International, Inc. (Temecula, CA, USA). A Rabbit ABC Elite kit (Vector Laboratories, Inc., Burlingame, CA, USA) was used to detect reactivities.
Histology and Immunohistochemistry
The pup heads were serially cut at 7 µm, deparaffinized, and stained with hematoxylin-eosin. The presence of enamel matrix was examined in a total of 73 upper first and second molars. Twenty-five teeth were from 8 pups of 4 control dams, 18 teeth were from 6 pups whose dams (3) were given 50 µg/kg TCDD, and 30 teeth were from 9 pups whose dams (3) were given 1000 µg/kg TCDD (Table
). The same examiner (A.K.) measured thicknesses of predentin and the whole dentin in digital images of 33 upper first and second molars (Pn22 rats) at the deepest site between the mesial and central (first molar) or the central and distal (second molar) principal cusps. The figures obtained were used for the calculation of predentin:whole-dentin ratios. Seven teeth were from 3 pups of 3 control dams. Sixteen teeth were from 5 pups whose dams (3) were exposed to 50 µg/kg TCDD, and 10 teeth were from 3 pups whose dams (2) were exposed to 1000 µg/kg TCDD (Table).
Twelve upper first and second molars were immunostained for AhR and CYP1A1. Four teeth were from 2 pups (1 Pn9, 1 Pn22) of a control dam, 4 teeth were from 2 pups (1 Pn9, 1 Pn22) whose dam received 50 µg/kg TCDD, and 4 teeth were from 2 pups (1 Pn9, 1 Pn22) whose dam received 1000 µg/kg TCDD (Table). As a slight modification to the manufacturer’s staining instructions, the rehydrated, rinsed sections were autoclaved for 20 min in 10 mM sodium citrate (pH 6.0), to improve staining. Endogenous peroxidase activity was quenched with 0.5% H2O2 in cold methanol. Primary antibodies to AhR and CYP1A1 were used at dilutions of 1:250 and 1:1000, respectively. Goat anti-rabbit IgG was used as the secondary antibody. The color reaction was made visible with AEC, and the sections were counterstained with hematoxylin. Reactions performed in the absence of primary antibodies were negative (not shown).
Statistics
The predentin:whole-dentin ratios at different exposure levels were compared by a chi-square test. The probability value of 
0.05 was considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
–1C) but was partly preserved at both the lower (Figs. 1D–1F) and higher (Figs. 1G–1I) (arrows) exposure levels in all teeth studied. Consistent with the gradient of enamel maturation, the matrix was most abundant cervically but was also retained in the erupted part of the first molars (Figs. 1J, 1K). Coronal predentin was thin in control rat teeth (Figs. 1A–1C). The predentin in the molars of the exposed pups appeared thicker, and its border to mineralized dentin was markedly globular (Figs. 1D–1I) (asterisks). The proportional thickness correlated with the exposure level (p < 0.001).
|
, 2C, 2E). Staining for CYP1A1 was strong in both cell types (Figs. 2B, 2D, 2F). TCDD did not alter the expression of either protein (Fig. 2) in any of the teeth analyzed.
|
, 3B). At the lower dose (dam given 50 µg/kg), TCDD clearly decreased AhR reactivity in ameloblasts and odontoblasts but did not change staining of the papillary layer (Figs. 3C, 3D). At the higher dose (dam given 1000 µg/kg TCDD), staining for AhR was barely detectable in ameloblasts and odontoblasts but was still visible in the papillary layer (Figs. 3E, 3F). Control rat ameloblasts and odontoblasts strongly expressed CYP1A1 (Figs. 3G, 3H), but the expression was weak at the lower dose of TCDD (Figs. 3I, 3J). At the higher dose, CYP1A1 expression was detectable in neither ameloblasts nor odontoblasts (Figs. 3K, 3L).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Enamel mineralization is a matrix- and cell-mediated process, which involves both secretion and degradation of the enamel matrix. The matrix provides the framework for the assembly and growth of apatite crystals (Fincham et al., 1999). However, the removal of matrix constituents by proteinases is necessary for the mineralization to be completed (DenBesten et al., 1989). Of the two major classes of proteolytic enzymes present in the developing enamel, metallo-endoproteinases predominate during the secretory stage and serine proteinases during the maturation stage (Robinson et al, 1998, and references therein). Hence, the metalloproteinases could be directly involved in the degradation of enamel matrix proteins, and the function of serine proteinases could be related to their removal. The sparse organic matrix of mature enamel is easily disintegrated and lost upon demineralization. With tooth eruption, ameloblasts undergo morphological changes and subsequently disappear. Therefore, abnormal processing of the matrix during the maturation stage may cause permanent mineralization defects (Paine et al., 2000). At an early stage of first and second molar development (Pn9), TCDD had no detectable effect on the retention of enamel matrix, obviously because of the high proportion of organic constituents in the immature enamel. However, the enamel matrix was completely degraded and lost in the control rat teeth by Pn22 but was partly retained in the exposed rat teeth. In line with our clinical findings that dioxins and dioxin-like compounds may be one causative factor of enamel mineralization defects in children via their mothers’ milk (Alaluusua et al., 1999), this suggests that TCDD arrests the degradation and/or removal of enamel matrix proteins.
Predentin exists throughout the lifetime of the tooth, which implies that the conversion of predentin to dentin involves not only cellular activities but also structural and compositional changes in the collagenous matrix (Beniash et al., 2000). Whether the retardation by TCDD of the mineralization of rat molars involves failure of implementation of mineralization-promoting mechanisms or of withdrawal of inhibitory factors is unknown.
Consistent with our previous findings of AhR expression in secretory dental cells of the mouse (Sahlberg et al., 2002), immunoreactivity of AhR was strong in control rat ameloblasts and odontoblasts. In contrast to the generally low CYP1A1 expression in unexposed animals, at least in the liver (Franc et al., 2001), control rat ameloblasts and odontoblasts expressed CYP1A1 clearly. In general, transient suppression of AhR after TCDD exposure is followed by up-regulation of AhR and CYP1A1 in vivo (Franc et al., 2001). However, the expression levels of AhR and CYP1A1 in ameloblasts and odontoblasts decreased with the increasing dose. At Pn9, exposure of the lactating dam to either dose of TCDD showed no obvious effects on the expression levels in the pup teeth. At Pn22, the initially high reactivities decreased sharply with the increase of the dose, concomitantly with the retention of excess enamel matrix after removal of the mineral phase. These results suggest that the depletion of AhR and CYP1A1 by TCDD is developmental stage- (or time-) and dose-dependent. They are also in line with results obtained by Western blottings, showing that TCDD reduces the AhR protein level in various tissues such as liver, spleen, thymus, lung, and reproductive organs (Pollenz et al., 1998; Roman and Peterson, 1998). Recent in vitro studies also show that TCDD induces AhR nuclear translocation and concomitantly promotes ubiquitin-mediated AhR degradation (Ma et al., 2000). It is well-documented that TCDD toxicity is, for the most part, mediated by AhR (Fernandez-Salguero et al., 1996), and also that the toxicity is dose-dependent. Interestingly, TCDD exposure depleted AhR reactivity in ameloblasts more quickly than in the papillary layer. Since the papillary layer appears at a late stage of tooth development (Shellis and Berkovitz, 1981), ameloblasts were available for exposure for a longer time, but there may also be sensitivity differences between secretory and non-secretory epithelial dental cells, leading to failure of the extinguished AhR to be up-regulated in secretory ameloblasts.
CYP1A1 gene expression is primarily induced by activators of AhR, and it is well-established that, upon TCDD binding, AhR translocates into the nucleus, where it induces CYP1A1 expression (Nebert et al., 2000). In H/W rats, CYP1A1 induction is sustained in the liver for at least 76 days after a single dose of TCDD (Pohjanvirta et al., 1990a), and increased expression in the liver is also found after lactational exposure (Iba et al., 2000). However, in our current study, TCDD dose-dependently reduced CYP1A1 expression in developing teeth. Recent studies have reported that chronic exposure of mouse to TCDD may cause a sustained oxidative stress response (Slezak et al., 2000), and this may repress the CYP1A1 gene transcription (Barouki and Morel, 2001). However, oxidative stress caused by TCDD in H/W rats is minimal, as measured by lipid peroxidation in the liver (Pohjanvirta et al., 1990b). Therefore, the dramatic reduction of CYP1A1 in odontoblasts and ameloblasts after TCDD exposure is more likely to be secondary to the repressed AhR expression and to be an early sign of impaired function of secretory dental cells.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
9 authors contributing equally to this work;
Received April 10, 2003; Last revision November 28, 2003; Accepted December 1, 2003
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alaluusua S, Lukinmaa PL, Torppa J, Tuomisto J, Vartiainen T (1999). Developing teeth as biomarker of dioxin exposure. Lancet 353:206.
Barouki R, Morel Y (2001). Repression of cytochrome P450 1A1 gene expression by oxidative stress: mechanisms and biological implications. Biochem Pharmacol 61:511–516.[ISI][Medline]
Beniash E, Traub W, Veis A, Weiner S (2000). A transmission electron microscope study using vitrified ice sections of predentin: structural changes in the dentin collagenous matrix prior to mineralization. J Struct Biol 132:212–225.[ISI][Medline]
DenBesten PK, Heffernan LM, Treadwell BV, Awbrey BJ (1989). The presence and possible functions of the matrix metalloproteinase collagenase activator protein in developing enamel matrix. Biochem J 264:917–920.[ISI][Medline]
Fernandez-Salguero PM, Hilbert DM, Rudikoff S, Ward JM, Gonzalez FJ (1996). Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicol Appl Pharmacol 140:173–179.[ISI][Medline]
Fincham AG, Moradian-Oldak J, Simmer JP (1999). The structural biology of the developing dental enamel matrix. J Struct Biol 126:270–299.[ISI][Medline]
Franc MA, Pohjanvirta R, Tuomisto J, Okey AB (2001). In vivo up-regulation of aryl hydrocarbon receptor expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a dioxin-resistant rat model. Biochem Pharmacol 62:1565–1578.[ISI][Medline]
Gu YZ, Hogenesch JB, Bradfield CA (2000). The PAS superfamily: sensors of environmental and developmental signals. Annu Rev Pharmacol Toxicol 40:519–561.[ISI][Medline]
Huuskonen H, Unkila M, Pohjanvirta R, Tuomisto J (1994). Developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the most TCDD-resistant and -susceptible rat strains. Toxicol Appl Pharmacol 124:174–180.[ISI][Medline]
Iba MM, Fung J, Cooper KR, Thomas PE, Wagner GC, Park Y (2000). Effect of gestational and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on the level and catalytic activities of hepatic microsomal CYP1A in prepubertal and adult rats. Biochem Pharmacol 59:1147–1154.[ISI][Medline]
Jan J, Vrbi V (2000). Polychlorinated biphenyls cause developmental enamel defects in children. Caries Res 34:469–473.[ISI][Medline]
Kattainen H, Tuukkanen J, Simanainen U, Tuomisto JT, Kovero O, Lukinmaa PL, et al. (2001). In utero/lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure impairs molar tooth development in rats. Toxicol Appl Pharmacol 174:216–224.[ISI][Medline]
Kiukkonen A, Viluksela M, Sahlberg C, Alaluusua S, Tuomisto JT, Tuomisto J, et al. (2002). Response of the incisor tooth to 2,3,7,8-tetrachlorodibenzo-p-dioxin in a dioxin-resistant and a dioxin-sensitive rat strain. Toxicol Sci 69:482–489.
Lukinmaa P-L, Sahlberg C, Leppäniemi A, Partanen A-M, Kovero O, Pohjanvirta R, et al. (2001). Arrest of rat molar tooth development by lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl Pharmacol 173:38–47.[ISI][Medline]
Ma Q, Renzelli AJ, Baldwin KT, Antonini JM (2000). Superinduction of CYP1A1 gene expression. Regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced degradation of Ah receptor by cycloheximide. J Biol Chem 275:12676–12683.
Nebert DW, Roe AL, Dieter MZ, Solis WA, Yang Y, Dalton TP (2000). Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochem Pharmacol 59:65–85.[ISI][Medline]
Paine ML, Zhu DH, Luo W, Bringas P Jr, Goldberg M, White SN, et al. (2000). Enamel biomineralization defects result from alterations to amelogenin self-assembly. J Struct Biol 132:191–200.[ISI][Medline]
Partanen A-M, Alaluusua S, Miettinen PJ, Thesleff I, Tuomisto J, Pohjanvirta R, et al. (1998). Epidermal growth factor receptor as a mediator of developmental toxicity of dioxin in mouse embryonic teeth. Lab Invest 78:1473–1481.[ISI][Medline]
Pohjanvirta R, Håkansson H, Juvonen R, Tuomisto J (1990a). Effects of TCDD on vitamin A status and liver microsomal enzyme activities in a TCDD-susceptible and a TCDD-resistant rat strain. Food Chem Toxicol 28:197–203.[ISI][Medline]
Pohjanvirta R, Sankari S, Kulju T, Naukkarinen A, Ylinen M, Tuomisto J (1990b). Studies on the role of lipid peroxidation in the acute toxicity of TCDD in rats. Pharmacol Toxicol 66:399–408.[ISI][Medline]
Pohjanvirta R, Wong JM, Li W, Harper PA, Tuomisto J, Okey AB (1998). Point mutation in intron sequence causes altered carboxyl-terminal structure in the aryl hydrocarbon receptor of the most 2,3,7,8-tetrachlorodibenzo-p-dioxin-resistant rat strain. Mol Pharmacol 54:86–93.
Pollenz RS, Santostefano MJ, Klett E, Richardson VM, Necela B, Birnbaum LS (1998). Female Sprague-Dawley rats exposed to a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibit sustained depletion of aryl hydrocarbon receptor protein in liver, spleen, thymus, and lung. Toxicol Sci 42:117–128.
Robinson C, Brookes SJ, Shore RC, Kirkham J (1998). The developing enamel matrix: nature and function. Eur J Oral Sci 106(Suppl 1):282–291.
Roman BL, Peterson RE (1998). In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs prostate development. 1. Effects on gene expression. Toxicol Appl Pharmacol 150:240–253.[ISI][Medline]
Sahlberg C, Pohjanvirta R, Gao Y, Alaluusua S, Tuomisto J, Lukinmaa P-L (2002). Expression of the mediators of dioxin toxicity, aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT), is developmentally regulated in mouse teeth. Int J Dev Biol 46:295–300.[ISI][Medline]
Schmidt JV, Bradfield CA (1996). Ah receptor signaling pathways. Annu Rev Cell Dev Biol 12:55–89.[ISI][Medline]
Shellis P, Berkovitz BKB (1981). Comparative anatomy of dentition. The dentition of laboratory rodents and lagomorphs. In: Dental anatomy and biology. Osborn JW, editor. Oxford: Blackwell Scientific Publications, pp. 432–439.
Slezak BP, Hatch GE, DeVito MJ, Diliberto JJ, Slade R, Crissman K, et al. (2000). Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol Sci 54:390–398.
Whitlock JP Jr (1999). Induction of cytochrome P4501A1. Annu Rev Pharmacol Toxicol 39:103–125.[ISI][Medline]
This article has been cited by other articles:
|
|
 |
|
  K. Yoshizawa, A. Heatherly, D. E. Malarkey, N. J. Walker, and A. Nyska A Critical Comparison of Murine Pathology and Epidemiological Data of TCDD, PCB126, and PeCDF Toxicol Pathol, December 1, 2007; 35(7): 865 - 879. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Sahlberg, E. Peltonen, P.-L. Lukinmaa, and S. Alaluusua Dioxin Alters Gene Expression in Mouse Embryonic Tooth Explants J. Dent. Res., July 1, 2007; 86(7): 600 - 605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Peltonen, P.-L. Lukinmaa, C. Sahlberg, A.-M. Partanen, A. Kiukkonen, and S. Alaluusua 7,12-Dimethylbenz[a]anthracene Interferes with the Development of Cultured Mouse Mandibular Molars Toxicol. Sci., July 1, 2006; 92(1): 279 - 285. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Miettinen, R. Sorvari, S. Alaluusua, M. Murtomaa, J. Tuukkanen, and M. Viluksela The Effect of Perinatal TCDD Exposure on Caries Susceptibility in Rats Toxicol. Sci., June 1, 2006; 91(2): 568 - 575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yoshizawa, N. J. Walker, M. P. Jokinen, A. E. Brix, D. M. Sells, T. Marsh, M. E. Wyde, D. Orzech, J. K. Haseman, and A. Nyska Gingival Carcinogenicity in Female Harlan Sprague-Dawley Rats following Two-Year Oral Treatment with 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Dioxin-Like Compounds Toxicol. Sci., January 1, 2005; 83(1): 64 - 77. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
