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Estrogen receptors in human pulp tissue
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Vol. 95 No. 3 March 2003
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY ENDODONTICS
Editor: Larz S. W. Spångberg
Estrogen receptors in human pulp tissue Silvana Jukic´, DDS, PhD,a Goranka Prpic´-Mehicˇic´, DDS, PhD,b Jasna Talan-Hranilovc´, MD, PhD,c Ivana Miletic´, DDS, PhD,d Sanja Sˇegovic´, DDS, PhD,e and Ivica Anic´, DDS, PhD,f Zagreb, Croatia UNIVERSITY OF ZAGREB AND CLINICAL HOSPITAL “SISTERS OF CHARITY”
Objective. The aim of this study was to evaluate the expression of estrogen receptor (ER)–␣ in human pulp tissue. Study design. Tissue samples were collected from 51 teeth extracted for orthodontic reasons or from endodontically treated teeth. Immunohistochemical staining was performed with mouse antihuman ER-␣ 1D5 by using the alkaline phosphatase antialkaline phosphatase technique. Immunohistochemical reactivity was semiquantitatively assessed and graded on an intensity scale of 0 to 3. Results. ERs were found in odontoblasts, endothelial cells, and Schwann cells. By means of statistical analysis, ERs were found to be common in odontoblasts (1.3-1.6) and in endothelial cells (1.0-1.4), whereas in Schwann cells they were somewhat rare (0.7-1.1). Difference in expression of ERs was not found among patients in different age groups. With respect to sex, a difference in reactivity was observed in Schwann cells, which showed higher reactivity in female pulps. Conclusion. The results of this study confirm the expression of ER-␣ in the human dental pulp. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:340-4)
Estrogen receptors (ERs) belong to the family of the ligand-activated transcription factors that regulate the growth, differentiation, and development of cells.1 Their function is to mediate the effects of the estrogen group of hormones on many tissues, most of them traditionally not considered as target tissues of these sex hormones.2 The formation of the hormone-receptor complex, interacting with chromosomal DNA, influences the transcription of target genes. Newly formed mRNA diffuses in cytoplasm and increases the produca
Assistant Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. b Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. c Professor, Clinical Department of Pathology “Ljudevit Jurak,” Clinical Hospital “Sisters of Charity,” Zagreb. d Assistant Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. e Assistant Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. f Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. Received for publication Mar 19, 2002; returned for revision Jun 24, 2002; accepted for publication Sep 12, 2002. © 2003, Mosby, Inc. 1079-2104/2003/$30.00 ⫹ 0 doi:10.1067/moe.2003.9
340
tion of proteins, changing the cell function. Immunohistochemical studies have revealed the presence of ER in the central nervous system, gastrointestinal tract, cardiovascular system, bone tissue, and immune system.3 In addition, some pathologic conditions associated with immune reactivity are more common in women than in men, especially during periods of dramatically changing levels of estrogen (eg, puberty, menopause, or pregnancy).4 Estrogen has been found to influence inflammation5 and the regulation of many tumors.6 The increased frequency of periodontal disease in women during a particular life period (eg, puberty, pregnancy, menstruation) or during the period of taking contraceptives has inspired scientists to examine oral— particularly periodontal—tissues with respect to the presence of ERs.7 Their findings were conflicting. Recently, however, a reverse transcriptase–polymerase chain reaction study revealed the existence of ER mRNA in periodontal ligament cells, although at low levels.8 ERs have been found in pyogenic granuloma in both males and females and pregnant and nonpregnant patients9 and in peripheral giant cell lesions of the jaws.10 Because of a report of multiple pulp polyps being more pronounced during the menstrual phase and be-
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 95, Number 3
Table I. Tissue samples grouped according to age and sex Age (y)
Female
Male
Total
⬍20 20-40 ⬎40 TOTAL
15 15 5 35
5 6 5 16
20 21 10 51
cause of the histologic similarity of the pulp polyps and pyogenic granuloma, the possibility of the presence of ERs in the pulp has been proposed.11 A variation in the level of estrogens throughout the lifespan could influence the physiologic and pathologic behavior of the pulp tissue. In their study of 20 human pulps, Whitaker et al11 did not find positive expression of ERs. However, the connective character of the pulp tissue, in that it has some elements that have already been proved to express ERs in other tissues,12,13 suggests the need for a more thorough examination. The aim of this study was to use the immunohistochemical method to evaluate the expression of ER ␣ in the human pulp tissue and the differences in occurrence in male and female patients during 3 periods of life. Female patients were also evaluated to determine the differences in ER expression in the dental pulp with respect to the phase of menstrual cycle. MATERIAL AND METHODS Pulp tissue samples were collected from teeth extracted for orthodontic reasons and from teeth endodontically treated as part of prosthetic therapy. Informed consent according to Helsinki II was obtained from each patient before investigation began. Patients were grouped according age and sex (Table I). Each group of women contained 3 subgroups divided in terms of menstrual cycles of equal duration. Of the 51 teeth, 40 were extracted for orthodontic reasons. The teeth extracted for orthodontic reasons were caries-free without any fillings or periodontal disease and were from otherwise healthy patients. Eleven teeth were extirpated for the purpose of prosthetic therapy. These teeth were also without caries profunda and periodontal disease. After extirpation with Hedstro¨ m files (Dentsply-Maillefer, Balaigues, Switzerland), the tissue was placed in 10% buffered formalin. The apices of the extracted teeth were resected to allow the fixative to penetrate into the pulp. Before being embedded in paraffin, the teeth were split and the pulps were gently removed. Paraffin sections were stained by means of the standard hematoxylin and eosin method. Immunohistochemical staining was performed by
Jukic´ et al 341
means of the alkaline phosphatase antialkaline phosphatase technique. Five-millimeter-thick tissue sections were placed on poly-L-lysine– coated slides and deparaffinized in xylene. After being placed in a plastic jar filled with 10 mmol/L citrate buffer (pH 6.0), the deparaffinized tissue sections were treated in a microwave oven 2 times for 5 minutes each time to allow the buffer to boil. Then the slides were left at room temperature in the buffer for approximately 20 minutes. After rinses with phosphate-buffered saline (PBS) solution, the slides were treated with primary monoclonal antibody (Dako-ER, 1D5; Dako, Glostrup, Denmark), which reacts with ER-␣,14 and incubated for 30 minutes at room temperature. After that, the specimens were again rinsed with PBS, placed in a buffer, and treated with secondary link antibody (biotinylated antirabbit and antimouse immunoglobulins in PBS; Dako) for 30 minutes at room temperature. The slides were rinsed with PBS buffer and treated with alkaline phosphatase antialkaline phosphatase complex and again rinsed with PBS solution. A chromogen-substrate solution (naphthol AS-MX [3-hydroxy-2-naphthoic acid 2, 4 dimethylanilide] with FAST blue Basic Base [4-amino-2, 5-dietholxybenzanilide] in 0.2 mol/L Tris buffer of pH 8.7) was added and incubated for 20 minutes at room temperature. Sections were then washed with water, counterstained with hematoxylin for 2 minutes, and dried. The sections were coverslipped with Dako Glycergel Mounting Media. The positive control was breast carcinoma tissue subjected to the same protocols as the study sample. For the negative control, mouse serum was substituted for the primary antibody. The immunohistochemical reactivities were semiquantitatively assessed by the interobserver consensus of 2 reviewers blinded to the clinical diagnosis (S.J., J.T-H.). The immunoreactivity was graded on an intensity scale of 0 to 3 (0, no reactivity; 1, 25% positive cells; 2, 50% positive cells; and 3, more than 50% positive cells). The statistical analysis was performed by using descriptive statistics and analysis of variance (P ⬍ .05). Intraexaminer reproducibility was studied by reassessing 30 randomly selected samples 1 month after the initial examination. There was 87.5% agreement in scoring. RESULTS Positive controls revealed nuclear ER reactivity. Immunohistochemical analysis of the human pulp samples revealed expression of ERs of nuclear localization in odontoblasts, endothelial cells, and Schwann cells (Figs 1 and 2). These structures demonstrated positive reactions in varying intensity from no reaction (grade 0) to an intensity approaching that of the positive control
342 Jukic´ et al
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY March 2003
Fig 1. The pulp from a 23-year-old woman. Odontoblasts (a), Schwann cells (b), and endothelial cells (c) positive for estrogen receptors (original magnification ⫻200).
Fig 3. The pulp from a 45-year-old man with 50% ERpositive odontoblasts.
Fig 2. The pulp from a 26-year-old man. ER-positive endothelial cells of the blood vessels (arrowheads) are seen (original magnification ⫻400).
Fig 4. The pulp from a 30-year-old man with 25% ERpositive odontoblasts.
(grade 3) (Figs 3 and 4). The mean expression of ERs in odontoblasts was calculated from sample results as 1.52 and, according to the intensity scale, could be expected to range in the population from 1.3 to 1.7. The mean expression in endothelial cells was 1.18 and ranged from 1.0 to 1.4, and the mean expression in Schwann cells was 0.9 and ranged from 0.7 to 1.1. No difference in the expression of ERs was found among patients of different age groups (Table II). The pulps of female patients had greater ER reactions in Schwann cells than did the pulps of male patients (P ⫽ .04; Table III). With respect to the phase of the menstrual cycle (Table IV), only Schwann cells showed the difference. Post hoc testing revealed that ER reactivity was expressed more in the second phase of the menstrual cycle than in the other phases and menopause (P ⬍ .05).
ical method. ERs were found in odontoblasts, endothelial cells, and Schwann cells. ERs in the pulp tissue were investigated by Whitaker et al,11 but the findings were negative. This is in contradiction to the results of our study, in which the expression of ERs was found in almost all dental pulps (male and female patients) with different activities. One of the explanations for this difference could be in the immunohistochemical methodology used and the relatively small number of samples in the previous study. The authors also had doubts about possible falsenegative results, claiming that 1 of the reasons for negative results could be the interaction of estrogens in the samples with ERs that hide their presence. A further reason for doubt is the presence of progesterone receptors in the same samples; it is well known that progesterone usually concentrates in estrogen-primed tissue.11 The findings of ERs in odontoblasts could be related to the expression of ERs in osteoblasts and osteoclasts,13 cells that produce or resorb mineralized tis-
DISCUSSION In this study, the expression of ERs in the human pulp was investigated by using the immunohistochem-
Jukic´ et al 343
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 95, Number 3
Table II. Means and SDs of estrogen receptor expression by age Odontoblasts
Endothelial cells
Schwann cells
Age (y)
Mean
SD
Mean
SD
Mean
SD
⬍20 20-40 ⬎40 All groups
1.60 1.62 1.20 1.53
0.60 0.80 0.63 0.70
1.15 1.19 1.20 1.18
0.59 0.75 0.63 0.65
0.75 1.09 0.80 0.90
0.72 0.94 0.63 0.80
Table III. Means and SDs of estrogen receptor expression by sex Odontoblasts
M F All groups
Endothelial cells
Nerve fibers
Means
SD
Means
SD
Means
SD
N
1.37 1.60 1.53
0.62 0.73 0.70
1.19 1.17 1.18
0.65 0.66 0.65
0.56 1.05 0.90
0.51 0.87 0.81
16 35 51
Table IV. Means and SDs of estrogen receptors by the phase of the menstrual cycle Odontoblasts
I II III Menopause All groups
Endothelial cells
Schwann cells
Means
SD
Means
SD
Means
SD
N
1.80 1.60 1.60 1.20 1.60
0.63 0.84 0.70 0.84 0.74
1.00 1.60 0.90 1.20 1.17
0.47 0.52 0.74 0.84 0.67
0.80 1.70 0.60 1.20 1.06
0.92 0.67 0.84 0.45 0.87
10 10 10 5 35
sue, where the role of ERs is to mediate the antiresorptive effects on bone.15 Given that ER expression is demonstrated in vascular tissue,12 their presence in the pulp endothelial cells is not surprising. Also, the neural tissue has been discovered to be ER-positive, and it has been shown that estrogens regulate the development and function of some parts of the central nervous system.3 ER expression in Schwann cells of the pulp is in accordance with findings of these receptors in Schwann-like macroglia cells in the central nervous system.16 The difference in ER expression found in Schwann cells in the pulp is not clear, because this is the only cell type that showed a statistically significant difference in distribution between sexes. A possible explanation is that some pulp samples could have been damaged by extirpation and some parts that contain the nerve tissue could have been missed. Because the sample of this study included only a small number of pulps taken from patients over 40 years of age, the lack of differences among the age groups and between men and women may be attributable to the low statistical power of such a comparison. The age group of patients over 40 years was small
because of difficult collection. Furthermore, a majority of the pulp samples were from orthodontically treated patients, which only rarely includes the elderly. Further studies are needed to clarify this issue. Today there is concern about the estrogenic activity of many environmental substances such as phytestrogens17 or phthalate esters that are widely used in many industrial products.18 The binding of such molecules to ERs could stimulate the expression of cellular estrogensensitive endpoints, genes, and mitosis. It has recently been reported that resins, materials used for esthetic results in cavity fillings, may provoke estrogenic activity in cells containing ERs.19 The main components responsible for such activity are bisphenol A and bisphenol-A dimethacrylate. It has been proved that in situ, polymerization is not completed and free monomers can be detected by using various analytic methods. The leaching of bisphenol-A components after the setting of resins, in concentrations that could evoke biologic effects, has been demonstrated.20 Depending on the depth of the preparation, the age of the patient, and the rate of tubular sclerosis, dentinal tubules are the most certain way of communication to the pulp. Direct
344 Jukic´ et al
placement of resin materials on the pulp tissue is also being researched.21 Bisphenol-A and related aromatic compounds are found in the saliva of patients during the 1-hour period after a resin-based fissure sealant is applied.18 Given that some salivary tumors express positive ERs6 and that periodontal tissue is also ERpositive,8 further research should be undertaken to establish the consequences of the usage of resin-based materials in dentistry. ER-␣ was found to be present in the pulp tissue. What remains to be further evaluated is whether the changes in hormone levels during various phases of the menstrual cycle or certain years could influence the physiologic and pathologic appearance of ER-␣ in the human pulp tissue and whether the placement of resin fillings, especially without a base formed by using the all-etch technique, influences pulp tissue because of the reaction with ERs. To our knowledge, this is the first study that reveals that ER-␣ is expressed in the dental pulp. Consequent events, their meaning, and significance should be investigated further. REFERENCES 1. Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988;240:889-95. 2. Sharpe RM. The role of estrogen in the male [review]. Trends Endocrinol Metab 1998;9:371-7. 3. Enmark E, Gustafsson JA. Estrogen receptors—an overview. J Intern Med 1999;246:133-8. 4. Holmdahl R. Female preponderance for development of arthritis in rats is influenced by both sex chromosomes and sex steroids. Scand J Immunol 1995;42:104-9. 5. Ushiyama T, Inoue K, Nishioka J. Expression of estrogen receptor–related protein (P29) and estradiol binding in human arthritic synovium. J Rheumatol 1995;22:421-6. 6. Jeannon JP, Soames JV, Bell H, Wilson JA. Immunohistochemical detection of estrogen and progesterone receptors in salivary tumors. Clin Otolaryngol 1999;24:52-4. 7. Parkar MH, Newman HN, Olsen I. Polymerase chain reaction analysis of estrogen and androgen receptor expression in human gingival and periodontal tissue. Arch Oral Biol 1996;41:979-83. 8. Morishita M, Shimazu A, Iwamoto Y. Analysis of estrogen receptor mRNA by reverse transcriptase–polymerase chain reaction in human periodontal ligament cells. Arch Oral Biol 1999; 44:781-83.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY March 2003 9. Whitaker SB, Bouquot JE, Alimario AE, Whitaker TJ Jr. Identification and semiquantification of estrogen and progesterone receptors in pyogenic granulomas of pregnancy. Oral Surg Oral Med Oral Pathol 1994;78:755-60. 10. Whitaker SB, Bouquot JE. Identification and semiquantification of estrogen and progesterone receptors in peripheral giant cell lesions of the jaws. J Periodontol 1994;65:280-3. 11. Whitaker SB, Singh BB, Weller RN, Bath KR, Loushine RJ. Sex hormone receptor status of the dental pulp and lesions of pulpal origin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:233-7. 12. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects of estrogen [review]. FASEB J 1996;10:615-24. 13. Oursler MJ, Landers JP, Riggs BL, Spelsberg TC. Estrogen effects on osteoblasts and osteoclasts. Ann Med 1993;25:361-71. 14. Pettersson K, Grandien K, Kuiper GGJM, Gustafsson J-Å. Mouse estrogen receptor  forms estrogen response element binding heterodimers with estrogen receptor ␣. Mol Endocrinol 1997;11:1486-96. 15. Ankrom MA, Patterson JA, Davis PY, Vetter UK, Blackman MR, Sponseller PD, et al. Age-related changes in human estrogen receptor alpha function and levels in osteoblasts. Biochem J 1998;333:787-94. 16. Gudino-Cabrera G. Nieto-Sampedro M. Estrogen receptor immunoreactivity in Schwann-like brain macroglia. J Neurobiol 1999;40:458-70. 17. Barnes S. Phytoestrogens and breast cancer. Baillieres Clin Endocrinol Metab 1998;12:559-79. 18. Moore NP. The estrogenic potential of the phthalate esters. Baillieres Clin Endocrinol Metab 1998;12:559-79. 19. Olea N, Pulgar R, Perez P, Olea-Serrano F, Rivas A, NovilloFertrell A, et al. Estrogenicity of resin-based composites and sealants used in dentistry. Environ Health Perspect 1996;104: 298-305. 20. Pulgar R, Olea-Serrano MF, Novillo-Fertrell A, Rivas A, Pazos P, Pedraza V, et al. Determination of bisphenol A and related aromatic compounds released from Bis-GMA– based composites and sealants by high performance liquid chromatography. Environ Health Perspect 2000;108:21-7. 21. Heitmann T, Unterbrink G. Direct pulp capping with dentinal adhesive resin system: a pilot study. Quintessence Int 1995;26: 765-70. Reprint requests: Silvana Jukic´ , DDS, PhD Department of Dental Pathology School of Dental Medicine University of Zagreb Gundulic´ eva 5, 10 000 Zagreb Croatia [email protected]
Vol. 95 No. 3 March 2003
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY ENDODONTICS
Editor: Larz S. W. Spångberg
Estrogen receptors in human pulp tissue Silvana Jukic´, DDS, PhD,a Goranka Prpic´-Mehicˇic´, DDS, PhD,b Jasna Talan-Hranilovc´, MD, PhD,c Ivana Miletic´, DDS, PhD,d Sanja Sˇegovic´, DDS, PhD,e and Ivica Anic´, DDS, PhD,f Zagreb, Croatia UNIVERSITY OF ZAGREB AND CLINICAL HOSPITAL “SISTERS OF CHARITY”
Objective. The aim of this study was to evaluate the expression of estrogen receptor (ER)–␣ in human pulp tissue. Study design. Tissue samples were collected from 51 teeth extracted for orthodontic reasons or from endodontically treated teeth. Immunohistochemical staining was performed with mouse antihuman ER-␣ 1D5 by using the alkaline phosphatase antialkaline phosphatase technique. Immunohistochemical reactivity was semiquantitatively assessed and graded on an intensity scale of 0 to 3. Results. ERs were found in odontoblasts, endothelial cells, and Schwann cells. By means of statistical analysis, ERs were found to be common in odontoblasts (1.3-1.6) and in endothelial cells (1.0-1.4), whereas in Schwann cells they were somewhat rare (0.7-1.1). Difference in expression of ERs was not found among patients in different age groups. With respect to sex, a difference in reactivity was observed in Schwann cells, which showed higher reactivity in female pulps. Conclusion. The results of this study confirm the expression of ER-␣ in the human dental pulp. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:340-4)
Estrogen receptors (ERs) belong to the family of the ligand-activated transcription factors that regulate the growth, differentiation, and development of cells.1 Their function is to mediate the effects of the estrogen group of hormones on many tissues, most of them traditionally not considered as target tissues of these sex hormones.2 The formation of the hormone-receptor complex, interacting with chromosomal DNA, influences the transcription of target genes. Newly formed mRNA diffuses in cytoplasm and increases the produca
Assistant Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. b Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. c Professor, Clinical Department of Pathology “Ljudevit Jurak,” Clinical Hospital “Sisters of Charity,” Zagreb. d Assistant Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. e Assistant Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. f Professor, Department of Endodontics, School of Dental Medicine, University of Zagreb. Received for publication Mar 19, 2002; returned for revision Jun 24, 2002; accepted for publication Sep 12, 2002. © 2003, Mosby, Inc. 1079-2104/2003/$30.00 ⫹ 0 doi:10.1067/moe.2003.9
340
tion of proteins, changing the cell function. Immunohistochemical studies have revealed the presence of ER in the central nervous system, gastrointestinal tract, cardiovascular system, bone tissue, and immune system.3 In addition, some pathologic conditions associated with immune reactivity are more common in women than in men, especially during periods of dramatically changing levels of estrogen (eg, puberty, menopause, or pregnancy).4 Estrogen has been found to influence inflammation5 and the regulation of many tumors.6 The increased frequency of periodontal disease in women during a particular life period (eg, puberty, pregnancy, menstruation) or during the period of taking contraceptives has inspired scientists to examine oral— particularly periodontal—tissues with respect to the presence of ERs.7 Their findings were conflicting. Recently, however, a reverse transcriptase–polymerase chain reaction study revealed the existence of ER mRNA in periodontal ligament cells, although at low levels.8 ERs have been found in pyogenic granuloma in both males and females and pregnant and nonpregnant patients9 and in peripheral giant cell lesions of the jaws.10 Because of a report of multiple pulp polyps being more pronounced during the menstrual phase and be-
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 95, Number 3
Table I. Tissue samples grouped according to age and sex Age (y)
Female
Male
Total
⬍20 20-40 ⬎40 TOTAL
15 15 5 35
5 6 5 16
20 21 10 51
cause of the histologic similarity of the pulp polyps and pyogenic granuloma, the possibility of the presence of ERs in the pulp has been proposed.11 A variation in the level of estrogens throughout the lifespan could influence the physiologic and pathologic behavior of the pulp tissue. In their study of 20 human pulps, Whitaker et al11 did not find positive expression of ERs. However, the connective character of the pulp tissue, in that it has some elements that have already been proved to express ERs in other tissues,12,13 suggests the need for a more thorough examination. The aim of this study was to use the immunohistochemical method to evaluate the expression of ER ␣ in the human pulp tissue and the differences in occurrence in male and female patients during 3 periods of life. Female patients were also evaluated to determine the differences in ER expression in the dental pulp with respect to the phase of menstrual cycle. MATERIAL AND METHODS Pulp tissue samples were collected from teeth extracted for orthodontic reasons and from teeth endodontically treated as part of prosthetic therapy. Informed consent according to Helsinki II was obtained from each patient before investigation began. Patients were grouped according age and sex (Table I). Each group of women contained 3 subgroups divided in terms of menstrual cycles of equal duration. Of the 51 teeth, 40 were extracted for orthodontic reasons. The teeth extracted for orthodontic reasons were caries-free without any fillings or periodontal disease and were from otherwise healthy patients. Eleven teeth were extirpated for the purpose of prosthetic therapy. These teeth were also without caries profunda and periodontal disease. After extirpation with Hedstro¨ m files (Dentsply-Maillefer, Balaigues, Switzerland), the tissue was placed in 10% buffered formalin. The apices of the extracted teeth were resected to allow the fixative to penetrate into the pulp. Before being embedded in paraffin, the teeth were split and the pulps were gently removed. Paraffin sections were stained by means of the standard hematoxylin and eosin method. Immunohistochemical staining was performed by
Jukic´ et al 341
means of the alkaline phosphatase antialkaline phosphatase technique. Five-millimeter-thick tissue sections were placed on poly-L-lysine– coated slides and deparaffinized in xylene. After being placed in a plastic jar filled with 10 mmol/L citrate buffer (pH 6.0), the deparaffinized tissue sections were treated in a microwave oven 2 times for 5 minutes each time to allow the buffer to boil. Then the slides were left at room temperature in the buffer for approximately 20 minutes. After rinses with phosphate-buffered saline (PBS) solution, the slides were treated with primary monoclonal antibody (Dako-ER, 1D5; Dako, Glostrup, Denmark), which reacts with ER-␣,14 and incubated for 30 minutes at room temperature. After that, the specimens were again rinsed with PBS, placed in a buffer, and treated with secondary link antibody (biotinylated antirabbit and antimouse immunoglobulins in PBS; Dako) for 30 minutes at room temperature. The slides were rinsed with PBS buffer and treated with alkaline phosphatase antialkaline phosphatase complex and again rinsed with PBS solution. A chromogen-substrate solution (naphthol AS-MX [3-hydroxy-2-naphthoic acid 2, 4 dimethylanilide] with FAST blue Basic Base [4-amino-2, 5-dietholxybenzanilide] in 0.2 mol/L Tris buffer of pH 8.7) was added and incubated for 20 minutes at room temperature. Sections were then washed with water, counterstained with hematoxylin for 2 minutes, and dried. The sections were coverslipped with Dako Glycergel Mounting Media. The positive control was breast carcinoma tissue subjected to the same protocols as the study sample. For the negative control, mouse serum was substituted for the primary antibody. The immunohistochemical reactivities were semiquantitatively assessed by the interobserver consensus of 2 reviewers blinded to the clinical diagnosis (S.J., J.T-H.). The immunoreactivity was graded on an intensity scale of 0 to 3 (0, no reactivity; 1, 25% positive cells; 2, 50% positive cells; and 3, more than 50% positive cells). The statistical analysis was performed by using descriptive statistics and analysis of variance (P ⬍ .05). Intraexaminer reproducibility was studied by reassessing 30 randomly selected samples 1 month after the initial examination. There was 87.5% agreement in scoring. RESULTS Positive controls revealed nuclear ER reactivity. Immunohistochemical analysis of the human pulp samples revealed expression of ERs of nuclear localization in odontoblasts, endothelial cells, and Schwann cells (Figs 1 and 2). These structures demonstrated positive reactions in varying intensity from no reaction (grade 0) to an intensity approaching that of the positive control
342 Jukic´ et al
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY March 2003
Fig 1. The pulp from a 23-year-old woman. Odontoblasts (a), Schwann cells (b), and endothelial cells (c) positive for estrogen receptors (original magnification ⫻200).
Fig 3. The pulp from a 45-year-old man with 50% ERpositive odontoblasts.
Fig 2. The pulp from a 26-year-old man. ER-positive endothelial cells of the blood vessels (arrowheads) are seen (original magnification ⫻400).
Fig 4. The pulp from a 30-year-old man with 25% ERpositive odontoblasts.
(grade 3) (Figs 3 and 4). The mean expression of ERs in odontoblasts was calculated from sample results as 1.52 and, according to the intensity scale, could be expected to range in the population from 1.3 to 1.7. The mean expression in endothelial cells was 1.18 and ranged from 1.0 to 1.4, and the mean expression in Schwann cells was 0.9 and ranged from 0.7 to 1.1. No difference in the expression of ERs was found among patients of different age groups (Table II). The pulps of female patients had greater ER reactions in Schwann cells than did the pulps of male patients (P ⫽ .04; Table III). With respect to the phase of the menstrual cycle (Table IV), only Schwann cells showed the difference. Post hoc testing revealed that ER reactivity was expressed more in the second phase of the menstrual cycle than in the other phases and menopause (P ⬍ .05).
ical method. ERs were found in odontoblasts, endothelial cells, and Schwann cells. ERs in the pulp tissue were investigated by Whitaker et al,11 but the findings were negative. This is in contradiction to the results of our study, in which the expression of ERs was found in almost all dental pulps (male and female patients) with different activities. One of the explanations for this difference could be in the immunohistochemical methodology used and the relatively small number of samples in the previous study. The authors also had doubts about possible falsenegative results, claiming that 1 of the reasons for negative results could be the interaction of estrogens in the samples with ERs that hide their presence. A further reason for doubt is the presence of progesterone receptors in the same samples; it is well known that progesterone usually concentrates in estrogen-primed tissue.11 The findings of ERs in odontoblasts could be related to the expression of ERs in osteoblasts and osteoclasts,13 cells that produce or resorb mineralized tis-
DISCUSSION In this study, the expression of ERs in the human pulp was investigated by using the immunohistochem-
Jukic´ et al 343
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 95, Number 3
Table II. Means and SDs of estrogen receptor expression by age Odontoblasts
Endothelial cells
Schwann cells
Age (y)
Mean
SD
Mean
SD
Mean
SD
⬍20 20-40 ⬎40 All groups
1.60 1.62 1.20 1.53
0.60 0.80 0.63 0.70
1.15 1.19 1.20 1.18
0.59 0.75 0.63 0.65
0.75 1.09 0.80 0.90
0.72 0.94 0.63 0.80
Table III. Means and SDs of estrogen receptor expression by sex Odontoblasts
M F All groups
Endothelial cells
Nerve fibers
Means
SD
Means
SD
Means
SD
N
1.37 1.60 1.53
0.62 0.73 0.70
1.19 1.17 1.18
0.65 0.66 0.65
0.56 1.05 0.90
0.51 0.87 0.81
16 35 51
Table IV. Means and SDs of estrogen receptors by the phase of the menstrual cycle Odontoblasts
I II III Menopause All groups
Endothelial cells
Schwann cells
Means
SD
Means
SD
Means
SD
N
1.80 1.60 1.60 1.20 1.60
0.63 0.84 0.70 0.84 0.74
1.00 1.60 0.90 1.20 1.17
0.47 0.52 0.74 0.84 0.67
0.80 1.70 0.60 1.20 1.06
0.92 0.67 0.84 0.45 0.87
10 10 10 5 35
sue, where the role of ERs is to mediate the antiresorptive effects on bone.15 Given that ER expression is demonstrated in vascular tissue,12 their presence in the pulp endothelial cells is not surprising. Also, the neural tissue has been discovered to be ER-positive, and it has been shown that estrogens regulate the development and function of some parts of the central nervous system.3 ER expression in Schwann cells of the pulp is in accordance with findings of these receptors in Schwann-like macroglia cells in the central nervous system.16 The difference in ER expression found in Schwann cells in the pulp is not clear, because this is the only cell type that showed a statistically significant difference in distribution between sexes. A possible explanation is that some pulp samples could have been damaged by extirpation and some parts that contain the nerve tissue could have been missed. Because the sample of this study included only a small number of pulps taken from patients over 40 years of age, the lack of differences among the age groups and between men and women may be attributable to the low statistical power of such a comparison. The age group of patients over 40 years was small
because of difficult collection. Furthermore, a majority of the pulp samples were from orthodontically treated patients, which only rarely includes the elderly. Further studies are needed to clarify this issue. Today there is concern about the estrogenic activity of many environmental substances such as phytestrogens17 or phthalate esters that are widely used in many industrial products.18 The binding of such molecules to ERs could stimulate the expression of cellular estrogensensitive endpoints, genes, and mitosis. It has recently been reported that resins, materials used for esthetic results in cavity fillings, may provoke estrogenic activity in cells containing ERs.19 The main components responsible for such activity are bisphenol A and bisphenol-A dimethacrylate. It has been proved that in situ, polymerization is not completed and free monomers can be detected by using various analytic methods. The leaching of bisphenol-A components after the setting of resins, in concentrations that could evoke biologic effects, has been demonstrated.20 Depending on the depth of the preparation, the age of the patient, and the rate of tubular sclerosis, dentinal tubules are the most certain way of communication to the pulp. Direct
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placement of resin materials on the pulp tissue is also being researched.21 Bisphenol-A and related aromatic compounds are found in the saliva of patients during the 1-hour period after a resin-based fissure sealant is applied.18 Given that some salivary tumors express positive ERs6 and that periodontal tissue is also ERpositive,8 further research should be undertaken to establish the consequences of the usage of resin-based materials in dentistry. ER-␣ was found to be present in the pulp tissue. What remains to be further evaluated is whether the changes in hormone levels during various phases of the menstrual cycle or certain years could influence the physiologic and pathologic appearance of ER-␣ in the human pulp tissue and whether the placement of resin fillings, especially without a base formed by using the all-etch technique, influences pulp tissue because of the reaction with ERs. To our knowledge, this is the first study that reveals that ER-␣ is expressed in the dental pulp. Consequent events, their meaning, and significance should be investigated further. REFERENCES 1. Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988;240:889-95. 2. Sharpe RM. The role of estrogen in the male [review]. Trends Endocrinol Metab 1998;9:371-7. 3. Enmark E, Gustafsson JA. Estrogen receptors—an overview. J Intern Med 1999;246:133-8. 4. Holmdahl R. Female preponderance for development of arthritis in rats is influenced by both sex chromosomes and sex steroids. Scand J Immunol 1995;42:104-9. 5. Ushiyama T, Inoue K, Nishioka J. Expression of estrogen receptor–related protein (P29) and estradiol binding in human arthritic synovium. J Rheumatol 1995;22:421-6. 6. Jeannon JP, Soames JV, Bell H, Wilson JA. Immunohistochemical detection of estrogen and progesterone receptors in salivary tumors. Clin Otolaryngol 1999;24:52-4. 7. Parkar MH, Newman HN, Olsen I. Polymerase chain reaction analysis of estrogen and androgen receptor expression in human gingival and periodontal tissue. Arch Oral Biol 1996;41:979-83. 8. Morishita M, Shimazu A, Iwamoto Y. Analysis of estrogen receptor mRNA by reverse transcriptase–polymerase chain reaction in human periodontal ligament cells. Arch Oral Biol 1999; 44:781-83.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY March 2003 9. Whitaker SB, Bouquot JE, Alimario AE, Whitaker TJ Jr. Identification and semiquantification of estrogen and progesterone receptors in pyogenic granulomas of pregnancy. Oral Surg Oral Med Oral Pathol 1994;78:755-60. 10. Whitaker SB, Bouquot JE. Identification and semiquantification of estrogen and progesterone receptors in peripheral giant cell lesions of the jaws. J Periodontol 1994;65:280-3. 11. Whitaker SB, Singh BB, Weller RN, Bath KR, Loushine RJ. Sex hormone receptor status of the dental pulp and lesions of pulpal origin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:233-7. 12. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects of estrogen [review]. FASEB J 1996;10:615-24. 13. Oursler MJ, Landers JP, Riggs BL, Spelsberg TC. Estrogen effects on osteoblasts and osteoclasts. Ann Med 1993;25:361-71. 14. Pettersson K, Grandien K, Kuiper GGJM, Gustafsson J-Å. Mouse estrogen receptor  forms estrogen response element binding heterodimers with estrogen receptor ␣. Mol Endocrinol 1997;11:1486-96. 15. Ankrom MA, Patterson JA, Davis PY, Vetter UK, Blackman MR, Sponseller PD, et al. Age-related changes in human estrogen receptor alpha function and levels in osteoblasts. Biochem J 1998;333:787-94. 16. Gudino-Cabrera G. Nieto-Sampedro M. Estrogen receptor immunoreactivity in Schwann-like brain macroglia. J Neurobiol 1999;40:458-70. 17. Barnes S. Phytoestrogens and breast cancer. Baillieres Clin Endocrinol Metab 1998;12:559-79. 18. Moore NP. The estrogenic potential of the phthalate esters. Baillieres Clin Endocrinol Metab 1998;12:559-79. 19. Olea N, Pulgar R, Perez P, Olea-Serrano F, Rivas A, NovilloFertrell A, et al. Estrogenicity of resin-based composites and sealants used in dentistry. Environ Health Perspect 1996;104: 298-305. 20. Pulgar R, Olea-Serrano MF, Novillo-Fertrell A, Rivas A, Pazos P, Pedraza V, et al. Determination of bisphenol A and related aromatic compounds released from Bis-GMA– based composites and sealants by high performance liquid chromatography. Environ Health Perspect 2000;108:21-7. 21. Heitmann T, Unterbrink G. Direct pulp capping with dentinal adhesive resin system: a pilot study. Quintessence Int 1995;26: 765-70. Reprint requests: Silvana Jukic´ , DDS, PhD Department of Dental Pathology School of Dental Medicine University of Zagreb Gundulic´ eva 5, 10 000 Zagreb Croatia [email protected]