ToxSci Advance Access originally published online on April 13, 2006
Toxicological Sciences 2006 92(1):279-285; doi:10.1093/toxsci/kfj199
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7,12-Dimethylbenz[a]anthracene Interferes with the Development of Cultured Mouse Mandibular Molars
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* Department of Pediatric and Preventive Dentistry and Department of Oral Pathology, Institute of Dentistry, 00014 University of Helsinki, Helsinki, Finland; and Department of Pathology and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland
1 To whom correspondence should be addressed at Biomedicum Helsinki, Institute of Dentistry, University of Helsinki, PO Box 63, FI-00014 Helsinki, Finland. Fax: +358 9 19125371. E-mail: eija.peltonen{at}helsinki.fi.
Received February 6, 2006; accepted April 10, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Clinical studies suggest that maternal smoking during pregnancy can reduce the crown size of the child's teeth. Delayed dental age compared with chronological age has also been reported in children whose parents smoke. Among the main components of tobacco smoke are nonhalogenated polycyclic aromatic hydrocarbons (PAHs), many of which are highly toxic. Humans are exposed to PAH compounds mainly via tobacco smoke and diet. The aim of our study was to investigate the effect of PAHs on tooth formation and the function of tooth-forming cells. We exposed mouse (NMRI) E18 mandibular first and second molar explants to 7,12-dimethylbenz[a]anthracene (DMBA), a toxic PAH compound, in organ culture for 7 or 12 days. DMBA concentrations used were 0.1, 0.5, 1, and 2µM. The mesiodistal width of each first molar (12-day culture) was measured in stereomicroscopic images, and the teeth were analysed histologically. DMBA exposure significantly reduced the mesiodistal width of the first molars. DMBA impaired or delayed amelogenesis and dentinogenesis in both molars at the lowest concentration of 0.1µM. DMBA affected enamel formation more severely than dentin formation and occasionally prevented amelogenesis completely. Elongation and polarization of ameloblasts were impaired, and blood vessel architecture of the dental papilla (future pulp) was altered. Cusps were thin and sharp. In line with the finding that maternal smoking during pregnancy has an adverse effect on child's tooth development, this study shows the toxic influence of PAHs on tooth development in vitro.
Key Words: polycyclic aromatic hydrocarbons; 7,12-dimethylbenz[a]anthracene; tooth development; mineralization; blood vessels; mouse.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Non-halogenated polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, toxic, environmental contaminants that are formed by incomplete combustion and carbonization processes of organic matter. Apart from some natural sources like smoke from forest fires and volcano eruptions, they are present in industry- and traffic-derived exhaust fumes, industrial by-products, and food. A number of different PAHs, such as benzo[a]pyrene (BP) and dibenz[a,h]anthracene, are one of the main toxic components of tobacco smoke (Hoffmann and Hecht, 1990
). Among nonsmokers, the principal route of PAH exposure is through the diet (Ramesh et al., 2004). The range of the dietary load of PAHs has been reported to be 0.02–17 µg/person/day (Ramesh et al., 2004). Tobacco smoke increases the daily PAH dose in an average of 0.1–0.25 µg per a nonfilter cigarette (Hoffman and Hecht, 1990). Hence, total PAH yield may be as high as 30 µg per day for heavy smokers (Menzie et al., 1992).
As demonstrated in animal experiments in vivo and in tissue and cell cultures in vitro, PAHs are cytotoxic, genotoxic, immunotoxic, teratogenic, and carcinogenic (Uno et al., 2004). The adverse effects of PAHs are thought to be mediated by the cytosolic aryl hydrocarbon receptor (AhR) (Gonzalez and Fernandez-Salguero, 1998). After binding of a ligand to AhR, the complex is translocated to the nucleus where it dimerizes with the AhR nuclear translocator (ARNT) protein. The ligand (e.g., PAH)-AhR-ARNT complex binds to specific DNA sequences and activates the transcription of certain genes. Of them, the best known are genes coding for xenobiotic-metabolizing enzymes such as cytochrome P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) (Shimada et al., 2003). CYP1A1 and CYP1B1 metabolize PAH compounds to active metabolites that are for the most part responsible for the toxic effects of PAHs (Gonzales, 2001).
The placenta protects the fetus by filtering noxious compounds from the maternal blood. Even so, the amount of BP metabolites bound to DNA as measured from placenta and umbilical cord blood was higher among smoking mothers than their nonsmoking counterparts (Arnould et al., 1997). Tooth development is sensitive to genetic aberrations and nongenetic factors such as environmental pollutants, which may irreversibly impair tooth formation. Clinical studies suggest that maternal smoking during pregnancy can reduce the crown size of the child's deciduous and permanent teeth (Heikkinen et al., 1992, 1994a,b). Accelerated clinical eruption and thinning of permanent incisors have also been reported (Heikkinen et al., 1995, 1997).
The basis of tooth development is inductive interactions between epithelial and mesenchymal cells and tissues (Thesleff, 2003). The first sign of tooth development is the thickening of oral epithelium at the site of a future tooth. In mice this occurs for the first time on embryonic day 11 (E11) when the mandibular first molars begin to develop. The epithelium forms a bud around which neural crest–derived mesenchymal cells condense (E12–E13). Morphogenesis continues through the cap (E14–E15) and early bell (E16) stages to the late bell stage (E18), at which the basic cuspal morphology has been completed. Differentiation of the ectomesenchymal odontoblasts and epithelium-derived ameloblasts and the subsequent deposition of dentin and enamel matrices, respectively, proceed from the tip of the mesial cusp in cervical and distal directions. Whereas mineralization of dentin does not start until a small amount of organic matrix has been laid down, mineralization of enamel begins coincidently with matrix deposition. The second molar passes through the various stages of development 2–3 days later than the first molar (Thesleff and Nieminen, 2001).
PAH compounds and polyhalogenated aromatic hydrocarbons such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) resemble each other chemically and share common mechanisms of action and general effects. The amount of PAHs in Finnish diet is three orders of magnitude greater than PCDD/Fs. With reference to the previous clinical observations of the adverse effect of tobacco smoke on developing human teeth, their sensitivity to PCDD/Fs (Alaluusua et al., 1999), and the experimental findings that developing rat and mouse teeth are responsive to the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Lukinmaa et al., 2001; Partanen et al., 1998, 2004), we studied the effects of dimethylbenz[a]anthracene (DMBA) on mouse embryonic molar tooth development in vitro.
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METHODS |
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Animals and teeth.
The embryonic age of mice (NMRI x NMRI) was set according to the day of the vaginal plug, which was designated day 0 (E0). The mice were anesthetized with CO2 and killed by cervical dislocation on E18 (Theiler Stage 26; Theiler, 1989
). We dissected 199 mandibular first and second molars from 101 pups of 12 dams. The explants were cultured in eight distinct experiments, once for 7 days and seven times for 12 days. A total of 24 teeth were cultured for 7 days and 173 teeth for 12 days. In each culture, there were 4–10 teeth in a single culture dish. A total of 78 explants (12 cultured for 7 days and 66 cultured for 12 days in eight experiments) were examined histologically, and 113 first molars from 63 pups representing six litters were measured for size comparison. These teeth had been cultured for 12 days in four experiments. The use of animals was approved by the Institutional Animal Care and Use Committee of the Faculty of Science of the University of Helsinki.
Organ culture.
Mandibular first and second molar tooth germs of E18 mice were dissected under a stereomicroscope and transferred onto polycarbonate Nuclepore filters (pore size, 0.1 µm; Corning Inc., New York, NY) on stainless steel grids placed in culture dishes (Trowell-type organ culture; Sahlberg et al., 2002a). The explants were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Paisley, Scotland) supplemented with 10% fetal calf serum (Gibco BRL) and ascorbic acid (100 µg/ml; Sigma, St Louis, MO) (basal medium). The DMBA congener 7,12-dimethylbenz[a]anthracene (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), commonly used to study toxic nonhalogenated PAHs, was added to the basal medium at the start of culture. DMBA was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10mM (DMBA stock). The working solution was made by diluting DMBA stock 1:100 in distilled water (DMBA concentration, 100µM). Final DMBA concentrations were 0.1, 0.5, 1, and 2µM. Control explants were cultured in the basal medium. DMSO may have undesirable cellular effects (Santos et al., 2003). In our previous studies, 0.7% (vol/vol) DMSO, used as a vehicle for TCDD, had no detectable effect on the development of mouse E18 molars cultured for up to 14 days (Partanen et al., 1998). In the present study, the concentration of DMSO was so low (< 0.04%) that any possible effects of DMSO could be ignored. The explants were cultured in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air for 7 or 12 days. The explants were inspected stereomicroscopically, and the medium was changed every 2–3 days. Those explants to be analyzed for the effect of DMBA on tooth size were cultured for 12 days.
Examination of the effect of DMBA on tooth size.
To study the effect of DMBA on the size of the mandibular first molar, tooth explants were digitally photographed under a stereomicroscope before further processing. The mesiodistal width of each tooth was measured at the level of the deepest site of the fissure distally to the principal mesial cusp using an analysis software (analySIS 3.00; Soft Imaging System GmbH, Münster, Germany). Differences in mesiodistal widths between control and DMBA-treated teeth were tested by Pearson's chi-square and Bonferroni's multiple comparison tests (SPSS, version 13, SPSS.com). The probability value of 
0.05 was considered significant.
Preparation of explants for histological examination.
After 7 or 12 days of culture, the explants were quickly fixed in cold methanol and fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4°C overnight. To facilitate sectioning of dental hard tissues, tooth germs were demineralized with 2.5% PFA + 12.5% ethylenediaminetetraacetic acid in PBS (a standard method for demineralization of calcified tissues) for at least 2 weeks, dehydrated, embedded in paraffin, serially sectioned at 7 µm, and stained with hematoxylin-eosin (HE). The results given are focused on changes seen after culturing tooth explants for 12 days since the effects became more clearly evident after 12 than after 7 days of culture.
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RESULTS |
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Stages of the First and Second Molar Development at the Start of Culture
As seen in stereomicroscopic examination of mouse E18 mandibular explants at the start of culture, morphogenesis of the three principal cusps of the first molar had been completed and the second molar was undergoing transition from the cap stage of morphogenesis to the bell stage (Fig. 1A). Histological examination of HE-stained serial sections showed that in the first molar ameloblasts were polarized throughout the tooth crown. Polarized odontoblasts facing ameloblasts were visible in the region of the mesial cusp and at the tip of the medial cusp but not in the distal cusp. Deposition of predentin had not started. The effect of DMBA on the first and second molar development was studied with special reference to the relationship of DMBA concentration to the differentiation and morphology of the dental cells and the dental hard tissue formation.
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The Effect of DMBA on the Size and Development of the Mandibular First Molar
The effect of 12 days of DMBA exposure on mandibular first molar size was studied by measuring the mesiodistal widths of the tooth crowns. The size decreased with the increasing DMBA concentration (p < 0.001, Pearson's chi-square test). Significant effect between the control and exposed tooth was first seen in explants cultured with 0.5µM DMBA (p < 0.02, Bonferroni's multiple comparison test). (See Figs. 1B–1F.)
After 12 days of culture, dentin mineralization and formation of the enamel in the unexposed control teeth had proceeded from the tip along the mesial side of the mesial cusp where it formed an even, continuous layer (Figs. 1B and 2A). The enamel was as thick as the unmineralized predentin and mineralized dentin together or even thicker (Fig. 2A). The distribution patterns of the enamel and mineralized dentin corresponded to each other, mineralized dentin consistently facing a layer of enamel. The extent of enamel and mineralized dentin varied from one culture experiment to another, ranging from the uniform presence in the mesial cusp to the distal cusp in occasional teeth. Consistent with the direction of dental cell differentiation and hard tissue formation, enamel and mineralized dentin were thickest at the mesial cusp tip and became thinner and more or less noncontinuous in the cervical and distal directions.
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After 12 days of culture with DMBA, morphology of the teeth was altered. The teeth were smaller than controls as depicted above, and their cusps were often thin and sharp (Figs. 1B–1F). Of the 10 first molar explants cultured with 0.1µM DMBA, two had a slightly thinner predentin than controls. The amount of enamel was normal in half of the teeth (Fig. 1C) and clearly reduced in the remaining half. Increasing DMBA concentration to 0.5µM did not increase the number of teeth with thinner predentin, but in the majority of teeth (8/11), the amount of enamel was reduced (Fig. 1D). Neither did it extend as far cervically or distally as in control teeth. Mineralization of dentin was retarded. In one explant of 11, neither mineralized dentin nor enamel was visible. In seven of 11 teeth, enamel was thinner than predentin and mineralized dentin together.
With the increase of DMBA concentration to 1µM and further to 2µM, the effects were aggravated and became evident in a higher proportion of teeth. Elongation of ameloblasts was less obvious than in controls (Figs. 2B and 2C), and part of the ameloblasts was completely nonpolarized as evidenced by the central location of the nuclei in the cytoplasms. The extent of enamel was more limited, and enamel thickness was reduced in all 17 teeth exposed to 1µM DMBA (Figs. 1E and 2B). One tooth was devoid of enamel. Of the 12 teeth exposed to 2µM DMBA, five lacked enamel, in three enamel was barely detectable (Figs. 1F, 2C, and inset in 2C), and in the remaining four the enamel layer was clearly thinner than in controls. When present, the enamel extended from the tip of the mesial cusp about midway along its mesial slope. In many teeth, enamel was restricted between the tip and the midpoint of the mesial cusp but was not present at the cusp tip (Figs. 1E and 2B). In the majority of explants, enamel was thinner than predentin and mineralized dentin together (Fig. 2B). Most teeth (14/17) exposed to 1µM DMBA and all 12 exposed to 2µM DMBA showed reduced predentin thickness (Figs. 2B and 2C).
Two of a total of 66 DMBA-exposed first molars, one cultured with 0.5 and the other with 1µM DMBA, showed an aberrant pattern of enamel matrix deposition. Enamel had in part been laid down on the basal aspect of ameloblasts. Furthermore, the enamel layer facing mineralized dentin was noncontinuous (data not shown).
Necrosis and/or apoptosis was visible both in controls and exposed teeth in the enamel organ between the mesial and medial cusps. Cell damage extended from the inner enamel epithelium to stellate reticulum and the outer enamel epithelium. In general, the extent of damaged area correlated with the size of the explant; necrosis and/or apoptosis was more frequent and pronounced in larger explants (data not shown).
The Effect of DMBA on Development of the Second Molar
After 12 days of culture, predentin formation in the unexposed control second molars was consistently in progress throughout the crowns (Figs. 1B and 2D). The thickness of predentin varied from one culture to another. Mineralized dentin facing enamel was visible in five of the 15 control teeth. In three of five teeth, enamel was visible only on the distal slope of the distal cusp (Fig. 2D), probably due to limitations in optimal nutrition centrally in the explant. Correspondingly, ameloblasts were mostly polarized and at least mesially and distally fully elongated. In the majority of teeth, mineralization of dentin or enamel formation had not started.
Consistent with the changes seen in the DMBA-exposed first molars, the exposed second molars were smaller than controls and their cusps were often thin and sharp (Figs. 1B–1F and 2D–2F). The effects were clear in teeth treated with 0.5µM DMBA (Fig. 1D), but already at the concentration of 0.1µM DMBA, morphology of half of the teeth was not perfectly normal. Ameloblasts were not consistently fully elongated, and predentin was slightly thinner than in control teeth. DMBA at the concentrations of 1 and 2µM had clearly affected most of the teeth with the severity of effects increasing with the concentration but varying between cultures. The thickness of predentin was reduced in 13 of the 15 teeth exposed to 1µM DMBA (Fig. 2E). In most second molars cultured with 2µM DMBA, only a thin layer of predentin was visible (Fig. 2F), and in one tooth predentin had not been deposited at all. Ameloblasts in most teeth had started to elongate (Fig. 2E), but in some they were largely nonpolarized as evidenced by the centrally located nuclei (Fig. 2F). Mineralization of dentin or enamel deposition had not started (Figs. 2E and 2F). Most teeth exposed to 2µM DMBA were extremely small and delicate.
The Effect of DMBA on Blood Vessel Development
DMBA also affected blood vessel development. The distribution pattern and size of blood vessels in the dental papilla (future pulp) were altered. In control teeth, small, delicate vessels were largely restricted to the mesial and medial parts of the papilla (Fig. 3A), whereas in DMBA-exposed teeth (1 and 2µM), thickish tubes containing occasional blood cells were more widely distributed (Figs. 3B and 3C). Large vascular spaces containing red blood cells were detected below first and second molars (Fig. 3D).
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DISCUSSION |
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In this in vitro study, we showed that DMBA, a representative of a class of toxic nonhalogenated PAHs, impaired dental hard tissue formation and reduced the size of cultured mouse E18 mandibular molar teeth dose and developmental stage dependently. In addition, blood vessel architecture of the dental papilla (future pulp) was altered.
PAH compounds are one of the main toxic components of tobacco smoke, and they are also found in an average diet (Hoffmann and Hecht, 1990; Ramesh et al., 2004). In clinical studies, tobacco smoke has been associated with size reduction of deciduous as well as permanent teeth (Heikkinen et al., 1992, 1994a,b). Delayed maturation of permanent teeth, that is, delayed dental age compared with chronological age has also been reported in children whose parents smoke (Kieser et al., 1996). Smoking increases the daily dietary intake of PAHs and further exposes the fetus to adverse effects of these compounds. Results of our experimental study support the clinical observations and indicate that DMBA both reduces tooth size and retards dental hard tissue formation and mineralization. On the other hand, the frequency of enamel defects in the permanent first molars (starting to develop at the 20th week of fetal life and mineralizing during the first years of life) was not found to be increased in children whose mothers had smoked during the last 12 months before delivery (Alaluusua et al., 1996).
Our study shows an in vitro effect of one tobacco smoke component on tooth development. Tobacco smoke also contains several other toxic compounds like nicotine, carbon monoxide, nitrogen oxides, and hydrogen cyanide (Hoffmann et al., 2001), which may have adverse effects of their own on tooth development. Nicotine has been shown to retard tooth development and to reduce the size of the first molar in rats and mice in vivo (Chowdhury and Bromage, 2000; Saad et al., 1991). In vitro, nicotine exposure caused necrosis in the dental papilla, and when this involved the odontoblast layer, the amount of predentin was reduced (Khan et al., 1981). Nicotine also impaired the secretion of enamel matrix (Khan et al., 1981). Thus, nicotine and PAH compounds seem to have similar effects that may act additively to impair tooth development.
The presence of the cytosolic AhR has been shown to be required for the toxicity of DMBA and other PAH compounds to become evident (Mann et al., 1999; Matikainen et al., 2001). We have previously shown that AhR and ARNT are coexpressed in developing tooth during the early stages of development and during the deposition of predentin and enamel (Sahlberg et al., 2002b). Hence, like the majority of adverse effects of dioxins (Gonzalez and Fernandez-Salguero, 1998), the detrimental effects of DMBA, even on tooth development, are likely to be mediated by the AhR-ARNT pathway. Our previous studies have shown that rodent teeth are dose and developmental stage dependently sensitive to the most toxic dioxin congener, TCDD, from the early morphogenetic stages until the completion of tooth formation (Kattainen et al., 2001; Lukinmaa et al., 2001; Miettinen et al., 2002; Partanen et al., 1998, 2004). Although DMBA and TCDD share the same receptor for induction of their noxious effects in different organs and tissues, their effects may differ. In the present study, we showed that the effects of DMBA on developing teeth resemble those of TCDD.
We firstly demonstrated that DMBA exposure reduces the size of cultured mouse mandibular first molar. This is in line with the findings that molar and incisor tooth size was reduced in rats exposed to TCDD (Alaluusua et al., 1993; Kattainen et al., 2001; Miettinen et al., 2002) and that treatment of developing mouse mandibular molars with TCDD in organ culture resulted in a smaller tooth size (Partanen et al., 2004). Secondly, we showed that DMBA impaired or delayed amelogenesis and dentinogenesis at a concentration as low as 0.1µM. TCDD also interfered with rat and mouse enamel and dentin formation in vivo and in vitro (Alaluusua et al., 1993; Gao et al., 2004; Partanen et al., 1998). Thirdly, we found that elongation of ameloblasts was delayed or they were nonpolarized, which had presumably led to an impaired function. The same effect was observed after TCDD exposure (Partanen et al., 1998). DMBA exposure also altered the cuspal morphology, and the effect was more severe in the second than in the first molar. Likewise, narrow and curved cusps were observed in mouse E18 first molars cultured for 11 days with TCDD (Partanen et al., 1998). Here we studied mouse E18 embryos whose second molars had already passed the early bud stage, which is critical for the continuation of tooth development. Therefore, development of the second molar was not arrested, which was the case when tooth germs of E14 embryos were exposed to TCDD (Partanen et al., 2004). However, at the highest DMBA concentration (2.0µM), one tooth was retained at the bell stage. If the same dose-response relationship is valid for TCDD and DMBA, DMBA exposure at earlier stages of tooth development is likely to have resulted in the arrest of the second molar development.
By contributing to the control of the number of cells and tissue modelling, apoptosis, programmed cell death, plays a central role in the normal development of various organ systems (Vaux and Korsmeyer, 1999). Developmental disorders and pathological processes can also involve apoptosis. In the course of normal tooth development, various cell types of the dental epithelium undergo apoptosis. From the cap stage to the early bell stage, apoptosis has been localized in the dental lamina. This epithelial cord connects the early tooth germ to the oral epithelium and is of vital importance to tooth development. The enamel knots, transient signaling centers that guide cuspal morphogenesis, disappear also apoptotically (Vaahtokari et al., 1996). The arrest of early mouse tooth development and cuspal deformation after dioxin exposure involved precocious and increased apoptosis but only in those dental epithelial cells that are normally predestined to go through a natural cell death, that is, the dental lamina and enamel knots, respectively (Partanen et al., 2004). Thus, the influence of TCDD on developing tooth is indirect and involves modification of gene expression. PAH compounds are known to induce the expression of an apoptosis-regulating gene, the bax gene (Matikainen et al., 2001, 2002). Accordingly, apoptosis induced by DMBA exposure of tooth explants may be one cause for tooth size reduction. PAHs also upregulate the expression of cytochrome P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) (Shimada et al., 2003), which are responsible for the generation of mutagenic metabolites and free radicals (Nebert et al., 2000). These may disturb the normal, intrinsic cell functions, which may lead to retarded tooth development and matrix formation. We observed dead cells both in controls and exposed explants, but specific staining methods differentiating between apoptosis and necrosis are required to study the effect of DMBA on apoptosis in cultured teeth. However, up to 25% of ameloblasts may die by apoptosis during normal development (Joseph et al., 1999), and interference with mouse tooth morphogenesis by TCDD involved enhanced apoptosis (Partanen et al., 2004). Therefore, reduction of tooth size after DMBA treatment, found here, could also have involved acceleration and/or increase of apoptotic death of ameloblasts.
Another potential cause of tooth size reduction is poor nutrition of dental pulp cells which may have led to impaired cell proliferation and, consequently, to smaller size of the tooth. Poor nutrition of odontoblasts may have resulted in retarded deposition and impaired mineralization of predentin. Since a prerequisite for the initiation of amelogenesis is mineralization of predentin, this may have secondarily impaired enamel deposition. Damage to vasculature has been proposed to be a key physiological mediator of the embryotoxicity of TCDD (Cantrell et al., 1996). DMBA has been shown to have angiogenic effects (Heffelfinger et al., 2000). In this study, we observed that DMBA may have a stimulating effect on the proliferation of pulpal blood vessels. However, the abnormal architecture of the vessels may impair their function. AhR has been found to play a role in the development of vasculature. A previous study has shown that adult AhR-deficient mice had fetal vascular structures resembling patent ductus venosus; they also had a reduced vascular architecture in kidneys (Lahvis et al., 2000). On the other hand, AhR stimulation by TCDD efficiently closed the patent ductus venosus in mice that had hypomorphic Ahr or Arnt allele and therefore reduced signaling of the AhR-ARNT pathway (Walisser et al., 2004). Deficient activation of AhR thus seems to retard development of the vasculature and overactivation appears to have an opposite effect. Since DMBA, like TCDD, activates AhR, it may have a similar effect on vasculature development as TCDD. Therefore, DMBA may have stimulated proliferation of endothelial cells and accelerated pulpal vasculature development.
In conclusion, we observed that DMBA interferes with the development of cultured mouse mandibular molars in different ways. The size of tooth germs was reduced, the cusps did not develop properly, and deposition and mineralization of the dental hard tissues were delayed. Blood vessel architecture was also changed. The effects were developmental stage and dose dependent with the sensitivity decreasing with advancing tooth development. This study confirms the toxic influence of nonhalogenated PAHs on tooth development and supports the earlier results of tooth size reduction caused by maternal smoking during pregnancy.
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ACKNOWLEDGMENTS |
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The skillful technical assistance of Ms Pirjo Jutila and Ms. Marjatta Kivekäs is gratefully acknowledged. The study was financially supported by the Academy of Finland (contract no. 206689) and the Finnish Dental Society Apollonia (E.P. personal grants).
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REFERENCES |
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