DEVELOPING TOOTH AS AN INDICATOR OF AND ORGAN MODEL FOR DIOXIN EXPOSURE

Project leader: Satu Alaluusua, University of Helsinki, Institute of Dentistry, P.O.Box 41, FIN-00014 University of Helsinki, Finland, tel. +358-9-19127314, e-mail: Satu.Alaluusua@helsinki.fi
 
 

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TIIVISTELMÄ SUOMEKSI

Researches:
IUniversity of Helsinki, e-mail: firstname.familyname@helsinki.fi
Pirjo-Liisa Lukinmaa, tel. +358-9-19127310
Carin Sahlberg, tel. +358-9-19125214
Anna-Maija Partanen, tel. +358-9-19125214
Anu Leppäniemi, tel. +358-9-19127304
Päivi Hölttä, tel. +358-9-19127359
Anu Kiukkonen, tel. +358-9-19125214
Outi Kovero, tel. +358-9-19127228
Collaborators:
National Public Health Institute, Kuopio, e-mail: firstname.familyname@ktl.fi: Terttu Vartiainen, Jouko Tuomisto, Raimo Pohjanvirta, Matti Viluksela,
Hannu Kiviranta, Hanna Kattainen, Jouni Tuomisto
National Public Health Institute, Helsinki: Jorma Torppa
Haartman Institute, University of Helsinki: Päivi Miettinen, e-mail: Paivi.Miettinen@helsinki.fi
Institute of Biotechnology, University of Helsinki: Irma Thesleff, e-mail: Irma.Thesleff@helsinki.fi

Consortium: Environmental health risk of dioxins
Financing SYTTY organisation: The Academy of Finland
Funding from SYTTY / Total funding of project (€): 104057 / 137695
Person-months of work funded by SYTTY / Total person-months of work: 61 / 72,4

KEY WORDS: dioxin, human milk, developmental dental defect, children
 

EXTENDED ABSTRACT

1 Introduction

Currently, tooth is one of the most extensively used organ model in developmental biology, in both health and disease. Firstly, determination of tooth form and the subsequent differentiation of tooth-specific cells result from epithelial-mesenchymal interactions, which are increasingly well known even at the molecular level. Also, tooth development is genetically determined but susceptible to external modification. Secondly, teeth can be cultured up to the stage of early mineralization and the development can be experimentally and selectively interfered with. Thirdly and most importantly, dental hard tissues, i.e. the enamel and the dentin, once they have been formed, are not remodelled. Hence, the consequences of any major disturbance in the function of the enamel-forming ameloblasts and the dentin-forming odontoblasts are permanent in nature.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals Dioxins pass placenta, accumulate in mother’s milk and are developmentally toxic. Their persistence and accumulation in tissue lipid and consequently, in the food chain, may result in chronic low-level exposure in humans. In 1993 we found that TCDD affects tooth morphogenesis and hard tissue formation in rats and three years later  that dioxins may cause enamel mineralization defects in children via their mothers’ milk.

The mechanisms underlying the spectrum of toxic responses elicited by TCDD are poorly understood. Several studies have indicated that the effects of TCDD are mediated by the aryl hydrocarbon receptor (AHR). Another pathway for developmentally toxic effects of TCDD may involve epidermal growth factor receptor (EGFR).

The general objectives of our studies were to show that dental defects serve as a biomarker of dioxin exposure and, that teeth can be used as an experimental organ model to determine the molecular background of the developmental toxicity of dioxins.

2 Methods

In this project we examined the dentitions of five different study groups/populations for developmental dental defects. Children included represented normal Finnish children who had been exposed to prevailing levels of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) and polychlorinated biphenyls (PCBs). In one study children who lived by the Kymijoki River in Kotka and Anjalankoski were examined. Recently, the sediments of the Kymijoki River were found to be heavily polluted by PCDDs and especially PCDFs. To examine the prevailing levels of PCDD/Fs and PCBs in human milk in the study area, milk samples from primiparae mothers were collected. Furthermore we determined the percentage of each PCDD/F congener of the sum of toxic PCDD/Fs to evaluate the contribution of individual congeners in the Kymijoki River sediments to the milk concentrations.

For these studies, milk samples were collected from the mother when the child was about 4 weeks old. The 17 most toxic PCDD/F and 36 PCB congeners were measured by gas chromatography-high resolution mass spectrometry in National Public Health Institute, Kuopio. The total exposure of the child was calculated from the concentrations of PCDD/F and PCB compounds in milk and the duration of breast feeding (Alaluusua et al. 1999). The permanent first molars, which are mineralized during the first two years of life, were chosen as target teeth and examined for hypomineralizations (Weerheijm et al., 2002).
Experimentally, we studied the effect of TCDD on the development of incisor and molar teeth of mice and rats in vivo and in an organ culture. Han/Wistar and Long-Evans rats were exposed to the concentrations varying from 0.03 to 1000 ?g/kg at different stages of tooth development. Embryonic molar teeth from EGFR deficient mice and embryonic and postnatal teeth from NMRI mice were studied in organ cultute. We determined the effects of TCDD on the expression of EGFR, epidermal growth factor (EGF), aryl hydrocarbon receptor (AHR), aryl hydrocarbon nuclear translocator protein (ARNT) and cytochrome P450 1A1 (CYP1A1) in tissue sections by histology, immunohistochemistry and RNA in situ hybridization

3 Results and discussion of the clinical studies

Developing teeth as a biomarker of dioxin exposure (Alaluusua et al., 1999). - We showed that mineralization defects occurred more often and were more severe in children who had been exposed to higher amounts of polychlorinated aromatic hydrocarbons than in those exposed to lower amounts. Notably, the defects were clearly associated with the total exposure to toxic dioxins and furans (p<0.004) but weakly with that to PCBs (p<0.07). Because the defects were seen after exposure to very low concentrations, we suggested that the dental defects were the best indicator of dioxin exposure available currently.

Hypomineralizations in the permanent first molars and their impact on the treatment need (Leppäniemi et al., 2001). - We showed that the prevalence of hypomineralization lesions in the permanent first molars of 7-13-year-old children living in Helsinki was about 19% (94/488). Dioxins may have caused a part of these lesions. 8.4% of the children had severe lesions which needed treatment or had already been treated. In 3.9% of the children all four molars were affected. The defects had significantly increased the treatment need of the target teeth (p<0.001). The results indicate that hypomineralizations are common and have a significant impact on treatment need in the current child population with low caries activity.

Developmental dental defects in children who resided by a river polluted by dioxins and furans (Hölttä et al., 2002). - Here we studied the possible influences of the polluted river Kymijoki on the riverside residents. Results showed that neither the levels of PCDD/Fs in human milk nor the prevalence of dental lesions were increased. Congeners dominating in the Kymijoki River sediments were not found in elevated levels in human milk. Furthermore, no correlation was found between the fish consumption of the mother and the dental lesions. In Anjalankoski but not in Kotka the duration of total breast-feeding was associated with the prevalence of the defects. Our results indicate that infants in the river area are not exposed to greater amounts of polychlorinated aromatic hydrocarbons than those living elsewhere in Finland.

4 Results and discussion of the experimental studies

EGFR as a mediator of developmental toxicity of dioxin in mouse embryonic teeth (Partanen et al. 1998). - To clarify whether epidermal growth factor receptor (EGFR), implicated in the mediation of the developmental toxicity of TCDD, is involved in the dental toxicity, we cultured embryonic molar teeth from EGFR deficient mice with TCDD, epidermal growth factor (EGF) and both agents in combination. In teeth of normal embryos, TCDD caused depolarization of odontoblasts and ameloblasts. Consequently, the dentin matrix failed to undergo mineralization and the enamel matrix was not deposited, and the cuspal morphology was disrupted. In teeth of the null mutant embryos, only the cuspal contour was mildly modified. EGF alone retarded the molar tooth development of normal embryos, but not that of EGFR deficient embryos. When coadministered with TCDD, EGF for the most part prevented the adverse effects of TCDD on teeth of normal but not EGFR deficient embryos. The result showed that the interference of TCDD with mouse molar tooth development in vitro involves EGFR signaling. Thus EGFR may also play a role in the developmental defects caused by dioxins in human teeth. Since EGFR is widely expressed in developing organs, EGFR signaling may even have general relevance in the mediation of the developmental toxicity of TCDD.

Arrest of rat molar tooth development by lactational exposure to TCDD (Lukinmaa et al., 2001 and unpublished results). - To study what developmental dental defects, if any, lactational exposure causes in rats, we gave lactating dams from the dioxin resistant Han/Wistar strain a high dose of TCDD (50 ?g or 1000 ?g/kg) one day after delivery. Of 19 animals studied histologically, 10 lacked one or more third molars. A higher proportion of pups exposed to the higher dose lacked third molars than of those exposed to the lower dose. The root tips of the more advanced first and second molars were prematurely closed and root formation was arrested. Dentinogenesis of the continuously erupting lower incisor teeth was preeruptively arrested because of odontoblastic cell death. All the teeth of the control rat pups developed normally. The results showed that the effect of TCDD on rat tooth development depend on not only the dose but also the tooth type and developmental stage. In asmuch as early tooth development is under the control of inductive interactions between the epithelium and the mesenchyme, the interference by TCDD with tooth morphogenesis with the consequent arrest of development is likely to involve epithelial-mesenchymal signaling.

To clarify the background of morphological consequences of TCDD exposure in teeth we analyzed the effect of TCDD on enamel and dentin mineralization and AHR and cytochrome P4501A1 (CYP1A1) expressions in amelobasts and odontoblasts. We could see retention of excess enamel matrix and thicker than normal predentin in the erupting molar (PnD22) of the exposed rats. With the increasing dose TCDD decreased the strong immunoreactivity of AHR and CYP1A1, visible in control rat ameloblasts and odontoblasts at PnD22. The results imply that interference by TCDD with tooth mineralization is AHR-dependent. This finding is in accordance with our findings in mice where we could see coexpression of AHR and ARNT during the formation and mineralization of the dental matrices by in situ hybridization and by immunohistochemistry.

In utero/lactational exposure to TCDD impairs molar tooth development in rats (Kattainen et al. 2000, 2001). - In this study we examined the effects of TCDD at low in utero/lactational exposure levels on rat tooth development in three rat lines that differed by their TCDD sensitivities and AHR structure. The results showed that TCDD at 1 ?g/kg completely prevented the development of the lower third molars in over half of the rats in the most sensitive rat line, while some pups in the more resistant lines were lacking this target tooth. The size of the molars was dose-dependently reduced in all rat lines. The lower third molars were most severely affected, and reduction was significant already at 0.03 ?g/kg in the most sensitive line. The results indicate that impaired tooth development is one of the most sensitive endpoints of TCDD-induced toxicity.

5 Conclusions

Our results show that developing teeth of different species are responsive to the adverse effects of dioxin. Since all developing organs utilize the same molecular signaling pathways and the dental hard tissues do not undergo remodelling, this makes  tooth an informative model for developmental toxicity of dioxins. In mice and rats exposure to TCDD via placenta or milk results in several developmental defects in developing teeth, the most severe of them being the arrest of early tooth germ development. The results suggest that the adverse effects of dioxin on tooth development is mediated not only via the EGFR pathway but also via the AHR/ARNT complex pathway. Whether these signaling pathways are coupled and whether other signaling pathways are present remains to be seen. In humans, the effects are seen at very low concentrations so that teeth may be the best available indicator of exposure at the population level. Notably, conclusions should not be extended to an individual. Because of the many well-established beneficial effects, and the declining dioxin levels in human milk breast-feeding should not be restricted on the basis of the possible dental effects.
 

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