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Abstract: The epigenetic nature of development mandates the observation of the effect of any exog- enous substance, especially those with estrogenic activities, during critical phases of development. The submandibular gland (SMG) presents as a great model due to extensive postnatal development, and is known to be regulated and affected by hormones as well as growth factors. Herein, we observed postnatal development following low doses of Biochanin A (BCA) and 17β estradiol (E2) in rats. The pups were randomly divided into four groups: control, BCA, E2, and dimethyl sulfoxide (DMSO), and euthanized at the 6th, 15th, 30th, and 60th postnatal days (PND). SMG morphogenesis was assessed. The nuclear expression of estrogen receptor beta (ERβ) was evaluated immu- nohistochemically; ERβ expression was up-regulated by BCA and down-regulated by E2. Similarly, caspase three gene expression, assessed by real time polymerase chain reaction was increased in the BCA group but decreased in the E2 group. A significant decrease in epidermal growth factor gene expression was noted at PND 30. The results presented by this study provide evidence that the effect of a postnatal exposure of the SMG to Biochanin A during devel- opment could be linked to sex hormone-dependent disorders.
Keywords: Biochanin A; 17β estradiol; dimethyl sulfoxide; submandibular salivary gland; morphogenesis; endocrinal disruptor.
Introduction Biochanin A (BCA, 7-dihydroxy-4’-methoxy 6 isofla- vone) is a phytoestrogen found in red clover, alfalfa, and cabbage (1). It is known for its various biological activities, which include protection of dopaminergic neurons, stimulation of osteoblastic cell differentiation, and inhibition of melanogenesis; in addition, it acts as an anti-proliferative and anti-inflammatory agent (2,3).
BCA is proposed to reverse, inhibit, or prevent several types of tumors possibly through the inhibition of several enzymatic activities as well as the induction of apoptosis (2,4,5). It has been shown to synergistically enhance the anti-proliferative and apoptotic effects of sorafenib (first line of treatment) in hepatocellular carcinoma cells (6). Furthermore, BCA is known for its estrogenic activity because it can bind with both alpha (ERα) and beta (ERβ) estrogen receptors (7).
Both ER isoforms belong to the family of nuclear receptors, a class of ligand-dependent transcription factors regulating the expression of genes contributing to growth, differentiation, and metabolism. In humans, the expression of ERα and ERβ are expressed in embryoid bodies as early as day 2, suggesting the role of estrogen in the differentiation and proliferation of human embry- onic stem cells, embryoid bodies, and DNA synthesis (8).
Estrogen regulates cellular responses by binding to ERs, thereby, regulating the transcription of target genes in the nucleus, and activating signaling pathways in the cytoplasm (9-12). 17β estradiol (E2) is the main contributor of the estrogen-dependent processes in peripheral tissues regulating bone growth and early
Journal of Oral Science, Vol. 59, No. 4, 579-588, 2017
Original
Effect of Biochanin A versus 17β estradiol in rat submandibular salivary gland
Amira M. Elsherbini, Mohammed A. R. Mohammed, and Fatma M. Ibrahim
Oral Biology Department, Faculty of Dentistry, Mansoura University, Mansoura, Egypt
(Received September 4, 2016; Accepted December 26, 2016)
Correspondence to Dr. Amira M. Elsherbini, Oral Biology Department, Faculty of Dentistry, Mansoura University, Elgomhoria Street, Mansoura 35516, Egypt E-mail: dramiraelsherbini2014@gmail.com J-STAGE Advance Publication: October 6, 2017 Color figures can be viewed in the online issue at J-STAGE. doi.org/10.2334/josnusd.16-0651 DN/JST.JSTAGE/josnusd/16-0651
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atherogenesis (13). Moreover, it is the main determinant for the proliferation of breast cancer cells in vivo (14,15). In an attempt to avoid the risks, the search for alternative therapy is justified, but the belief that “natural” alterna- tives have no adverse effect is mistaken (16).
Though considered as non-target tissue for estrogen, it was found that submandibular glands (SMG) contain a factor that can directly or indirectly modulate estrogen synthesis in the ovaries. SMG removal (sialoadenec- tomy) increased estrogen production and uterine weight; on the other hand, ovarian estrogen could be inhibited by the submandibular gland via inhibition of ovarian androgen production (17). Additionally, the expression of estrogen receptors (predominantly ERβ) is higher in the submandibular gland when compared with the other major salivary glands, thus implicating the role of sali- vary glands in the development and/or regeneration of reproductive organs and peripheral organs (16-18).
The epigenetic nature of development raised a lot of concern regarding exposure to exogenous compounds, especially endocrinal disruptors, due to their ability to alter the developmental trajectory during critical develop- mental periods at low concentrations. The parenchymal elements of rodent SMG exhibit extensive development during postnatal periods. In the present study, the prepu- bertal development of rat SMG following low doses of BCA and E2 exposure was carried out to experiment whether BCA is a safe natural product or merely an endocrinal disruptor. To the best of our knowledge, this is the first study to assess the effect of BCA on the post- natal development of SMG. The null hypothesis was that dimethyl sulfoxide and the controls are the same. BCA modulated postnatal growth in mammary glands and was therefore, expected to enhance SMG development (14).
Although BCA is thought to be better tolerated and has favorable protein expression effect than genistein (soy isoflavone) (14), the present study presented the damaging effects of BCA that may exceed those revealed by genistein on the submandibular gland.
Materials and Methods The study was carried out at the Oral Biology Department, Faculty of Dentistry, Mansoura University. All proce- dures performed in this study involving animals were in accordance with the ethical standards in compliance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised, 1978), and approved by the Ethics Committee of Faculty of Dentistry, Mansoura University, Egypt (No. 24805, 2013). All efforts were made to minimize animal suffering, and to reduce the number of animals used.
Materials E2 (E8875) and BCA (Sigma Aldrich Co., St. Louis, MO, USA), dimethyl sulfoxide (DMSO; Abbiotec, San Diego, CA, USA).
Study design Pregnant, female, pathogen-free Sprague Dawley rats were acclimatized under standardized temperature, humidity, and phytoestrogen-free diet conditions, and housed in individual polypropylene cages to avoid xeno- hormone residues.
After mating and parturition, 96 pathogen-free female offspring were randomly allocated into four groups (n = 24). Group 1 was the control group, whereas in the experimental groups, the offspring were subcutaneously injected with 500 μg/g body weight of BCA dissolved in an equal volume (1:1) of DMSO (group 2), 500 ng/g body weight of E2 dissolved in sesame oil (group 3), and corresponding volume of the DMSO (group 4) at post- natal days (PND) 1, 5, 14, 21, 30 targeting the various developmental stages (14,19).
Biopsy collection The offspring were euthanized on the 6th, 15th, 30th, and 60th PND (six on each day). The submandibular salivary glands were surgically removed on both sides and separated from the sublingual glands; the right half was processed for routine histological and immunohis- tochemical examinations, and the left half for molecular examination (20).
Histological and immunohistochemical examinations Specimens were fixed in 10% neutral buffered formalin, dehydrated, cleared, infiltrated, and subsequently embedded with paraffin wax. Sections (4 μm) were deparaffinized, hydrated, and stained with Meyer’s Haematoxylin and Eosin (HE) stain (Tissue Pro Tech- nology, Gainesville, FL, USA) (21).
Immunohistochemical staining After heat-induced epitope retrieval (22) and blocking of endogenous peroxidase activity, the samples were incubated with primary mouse monoclonal antibody ERβ (two drops or 100 µL; Dako Co., Santa Clara, CA, USA), biotinylated secondary antibody (Vector Labora- tories, Burlingam, CA, USA), and strepavidin peroxidase (Abcam, Cambridge, MA, USA), respectively.
The addition of DAB Substrate-Chromogen Solution (BioLegend, San Diego, CA, USA) resulted in brown deposits at the site of target antigen. Negative controls were run in parallel by replacing the primary antibody
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with non-immune immunoglobulins. Counter staining with hematoxylin was performed followed by mounting and examination of the slides under the light microscope.
Computer-assisted digital image analysis (Digital morphometric study) Slides were photographed using Mikroskop Olympus CX22 (Olympus, Tokyo, Japan) installed on an Olympus microscope with a 1/2 X photo adaptor, using 40 X objective. The resulting images were analyzed on Intel Core I3 based computer (Intel, Santa Clara, CA, USA) using Video Test Morphology software (Dvinskaya str., Saint Petersburg, Russia) with a specific built-in routine for % area measurement and automated object counting.
Five slides from each case were prepared and five random fields from each slide were analyzed. Micro- scopic analyses were performed by double-blinded calibrated examiners. After enhancing the color tones and contrasts of the acquired images in order to reveal the target stain color, thresholding of the image at the level of the desired hue range was performed to form a binary mask that represents the target cells; then each binary mask was defined as the region of interest (ROI).
Object counting routine was applied on each ROI using size and circularity filters to exclude artifact objects. The results were exported to an XLS sheet and percentage calculations of positively stained cells was calculated and tabulated.
Real-time polymerase chain reaction (RT-PCR) Total ribonucleic acid (RNA) was extracted from the collected SMG using GF-1 total RNA extraction kit (Vivantis Technologies Sdn Bhd, Selangor Darul Ehsan, Malaysia). The concentration and purity of each RNA sample was determined using a Nano-Drop Spectropho- tometer (Implen, München, Germany). The purity of the samples was validated by running them on an agarose gel. Pure samples were then reverse-transcribed to complementary DNA (cDNA) using Thermo Scientific Revert Aid First strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). For each
sample, 2 μL of the template cDNA was amplified with the Thermo Scientific Maxima SYBR Green qPCR kit (Thermo Fisher Scientific) using gene-specific primers for epidermal growth factor (EGF) and caspase-3 (Cas- 3). The primer sequences listed in Table 1 were obtained from Pubmed (Entrez Gene). Glyceraldehyde 3-phos- phate dehydrogenase (GAPDH) was used as reference (housekeeping) gene. The amplification and detection of targeted and housekeeping genes were performed in duplicate for each sample.
The thermo-cycler program consisted of an initial activation at 95°C for 10 min, followed by three-step cycling (40 cycles) starting with denaturation at 95°C for 15 s, annealing at 60°C for 10 s, and finally extension at 72°C for 1 min.
Calculation Relative quantification of mRNA expression was calcu- lated using the 2−ΔΔCt method. The data were presented as relative quantity of target mRNA, normalized against GAPDH mRNA and relative to a calibrator sample. Normal samples were used as calibrators, where,
ΔCt = (Ct of target gene – Ct of reference gene) ΔΔCt = (ΔCt of sample – ΔCt of normal) Ct is defined as the fractional cycle number at which
the fluorescence passes the fixed threshold.
Statistical analysis Data are presented as mean ± standard deviation (SD). Multiple comparisons were performed using one-way ANOVA followed by Tukey-Kramer’s post-hoc test. Differences between groups were considered significant at P < 0.05. All statistical analyses were performed using GraphPad Prism software package (version 6, La Jolla, CA, USA).
Results Hematoxylin and Eosin results Histological sections of the rat SMG stained with H and E in the control group revealed gradually expanding lobes and lobules, which replaced the transient structures (terminal tubules and proacini; Fig. 1) with mature forms of acini and duct systems. The granular convoluted ducts were observed at PND 60 (Fig. 2).
In the BCA and E2 groups, the glands showed increase in size with apparent increase in the number of acini and ducts at PND 6 and 15, along with transformation into acini and well-organized ducts and some cytoplasmic vacuolation (Fig. 1). Apparent vascularity was noticed in the E2 group. The increase in the number of acini and ducts was lower in the DMSO group when compared
Table 1 Primers used in real-time polymerase chain reaction Primer Sequence GAPDH Forward 5’-CCA GGG CTG CCT TCT CTT GT-3’
Reverse 5’-CTG TGC CGT TGA ACT TGC CG-3’ EGF Forward 5’-GGCCTCTGACTCCGAACGAT-3’
5’-GACAAAAGAAACCCGCCTG-3’ Reverse 5’-CCGACGAGTCTGAGTTGGGG-3’
Cas-3 Forward 5’-GGAGCTTGGAACGCGAAGAA-3’ Reverse 5’-CAGAGTCCATCGACTTGCTTCC-3’
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with the other groups (Fig. 1). Subsequenlty, both BCA and E2 groups revealed widely
spaced lobules with increase in both size and number of acini. The walls of the duct system showed some collapse with reduction in the resulting spaces around these ducts. In the DMSO group, there was a considerable amount of vacuolation in the acinar structures along with fibrosis around the ducts. By the 60th PND there was an increase in the amount of vacuolation; no granular convoluted ducts were bserved in all the experimental groups (Fig. 2).
Immunohistochemical results The presence of ERβ receptor in rodent SMG was revealed via mild cytoplasmic reactions in the proacini in the control, BCA, and E2 groups. At PND 15, the terminal tubules in the control group showed mild
cytoplasmic reactions. The reaction was negative in the other groups, except for the DMSO group, which showed strong cytoplasmic reactions. All groups showed a significant decrease in the cytoplasmic expression of ERβ when compared with the control group at PNDs 6 (BCA 41.8%; E2 90.7%; DMSO 24.6%; P < 0.0001) and 15 (BCA 27.2%; E2 89.5%; DMSO 69.9%; P < 0.0001). The decrease was significant both between the groups and in comparison to the control group as revealed statis- tically by the post-hoc Tukey’s multiple comparisons test (Table 2, Fig. 3).
The ERβ receptors were detected in the nuclei of SMG duct cells in all groups, and the cytoplasm of the cells in the DMSO group only. Except for mild reactions in the DMSO group, all groups revealed negative reactions in the acini. Tukey’s multiple comparisons test revealed significant differences between all experimental groups
Table 2 Tabulated means and standard deviations (SD) of the imunno-positive cytoplasmic results at PND 6 and 15 PND Control BCA E2 DMSO P value 6 PND 4.883 ± 0.01211 2.843 ± 0.001265 0.4563 ± 0.002066 3.682 ± 0.3327 P < 0.0001 15 PND 4.313 ± 0.00121 3.140 ± 0.01414 0.4548 ± 0.00147 1.298 ± 0.8150 P < 0.0001
Fig. 1 Photomicrograph of rat SMG stained with H&E at the 6th PND (A: Control; B: BCA; C: E2; and D: DMSO group). Magni- fication ×400. A: The glandular epithelium consisted of proacinar cells bulging from terminal tubules. B and C: BCA and E2 groups showed increased branching of terminal tubules with transforma- tion into acini and well-organized ductal structures similar to the striated duct (SD). D: The DMSO group showed decreased branching and wide interstitial spaces. TT, terminal tubules; PA, proacini; B, blood vessel; SD, striated duct; V, vacuoles.
Fig. 2 Photomicrograph of rat SMG stained with H&E at the 60th PND. A: Control; B: BCA; C: E2; and D: DMSO (Magni- fication ×400). In the control group, the acini were larger in size with differentiation of granular convoluted ducts (GCD). Note: There were no GCDs in all experimental groups. B: The striated duct showed changes in the wall with some collapse (*). C: Vacu- olation in the acinar structure. D: Increased vacuolation. GCD, granular convoluted ducts; BV, blood vessel; V, vacuolation; A, acini; SD, striated duct; S, spaces around the ducts.
Table 3 Tabulated means and standard deviations (SD) of imunno-positive nuclear results at PND 30 and 60 PND Control BCA E2 DMSO P value 30 PND 15.67 ± 3.615 7.970 ± 0.7981 4.150 ± 1.030 11.07 ± 0.8962 P < 0.0001 60 PND 20.66 ± 5.817 30.67 ± 2.73 7.976 ± 5.996 10.29 ± 3.907 P < 0.0001
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(except E2 and DMSO) and the control group. At PND 30, all groups demonstrated decreased nuclear reaction for ERβ when compared with the control group (BCA 49.1%; E2 73.5%; DMSO 29.4%; P < 0.0001). However, as seen in Figs. 3 and 4, a significant increase in nuclear reaction for ERβ was noted in the BCA group (48.5%) at PND 60, whereas, the other experimental groups presented with significant decrease in reactions to the receptor (E2 61.4%; DMSO 50.2 %; P < 0.0001; Table 3).
Real-time PCR results The results represent the means ± SD of Cas-3 and EGF genes calculated using the expression levels normalized to the levels in normal developing rats (Tables 4, 5).
The expression of Cas-3 was detected in all ages in the control group. There was a 1.695, 1.512, and 1.103 fold increase in the BCA group when compared with the control group at the 6th, 15th, and 60th PND, respec- tively. The E2 group demonstrated a 0.4268, 0.2710, and 0.5246 fold decrease, whereas the DMSO group showed a 0.2217, 0.1739, and 0.4530 fold decrease when compared with the control group at the 6th, 15th, and 60th PND, respectively. At the 30th PND, there were 0.5333, 0.2435, and 0.2006 fold decrease in gene expression in the BCA, E2, and DMSO groups, respectively. Tukey’s test revealed significant changes in all groups at different times when compared to the controls, except during the 30th PND (Fig. 5).
EGF expression was detected in all age groups in the control group and peaked at PND 60. A 0.6652 and 0.8537 fold decrease was noted in the BCA and E2 groups, respectively at PND 6, whereas at PND 15, a fold increase of 1.053 and 1.463 was observed in the two respective groups, when compared with the
Fig. 4 Immunohistochemical photomicrograph of ER-β (mag- nification ×400) nuclear expression in an SMG section from the control group at the 60th PND. The nuclear expression was observed only in the ductal component (arrows) while the acini showed no reaction (neither cytoplasmic nor nuclear).
Fig. 3 Effect of prepubertal exposure to 500 μg/g body weight (BW) of BCA, 500 ng/g BW of E2, and 500 μg/g BW of vehicle DMSO on ERβ expression results assessed by image analysis in all groups. Cytoplasmic expression of ERβ was observed at the 6th and 15th PND, while the nuclear expression of ER-β was revealed at the 30th and 60th PND. The values represent the means ± SD (n = 6). Intergroup differences (Tukey test) are indicated by the following symbols: *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; and ns, non-significant.
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controls. Moreover, a 1.224 and 1.095 fold increase was observed in the DMSO group at the 6th and 15th PND, respectively, whereas at the 30th PND, a fold decreas of 0.5065, 0.2166, and 0.1885 was noted in the BCA, E2, and DMSO groups, respectively. At the 60th PND, there was a 1.436, 1.379, and 1.240 fold increase in the BCA, E2, and DMSO groups respectively. Tukey’s multiple comparisons test revealed significant decrease in all groups when compared with the control group at PND 30, whereas, a non-significant decrease was noted at PND 6. Except for E2 at PND 15, non-significant increases were observed in all groups at PND 15 and 60 (Fig. 6).
Discussion The rodent SMG is considered a perfect model for studying postnatal development and growth. In the present study, the control group showed a gradual transformation from the proliferation phase to the differentiation phase, replacing transient structures such as terminal tubules with mature forms of the gland. The latter is accompanied by the appearance of granular convoluted ducts at the 60th PND. This is in accordance with the study by Cestari et al. (23) which reported that newborn rat submandibular glands present with transient secretory units, terminal tubules, during the first two weeks, which reduces by the end of the 3rd week. The late development of granular convoluted ducts (GCD) in females may be due to low levels of androgen, in addition to extrinsic factors such as body weight, hormones, and dietary changes associated with weaning, thus resulting in important differential growth changes (23,24).
In comparison to the control group, H&E stained sections from the BCA and E2 groups, but not the DMSO group revealed an increase in the number of acini and ducts. These results are partly in agreement with those
reported by Mishra et al. (14); similar concentrations of BCA and estradiol benzoate, a known estrogen agonist, were observed in the mammary gland along with enhanced proliferation of the mammary gland tubules and terminal end buds. On the other hand, contrary to the findings of Mishra et al. (14), wherein no differences in mammary gland differentiation were noted when compared to the controls in the DMSO group, a decrease in the same endpoint was observed in the DMSO group in the present study.
These results may be explained by the ability of BCA to induce proliferation at small concentrations lower than 0.1 to 10 μM, whereas a similar proliferative capacity of estradiol is achieved at 0.2 or 0.3 μM on MCF7 cells in vitro (25-28). Additionally, the results in the current study may be attributed to the effect of BCA and E2 on DNA synthesis. Concomitantly, Jefferson et al. (29) revealed that both BCA and daidzein (isoflavone) affect hormonal endpoints such as increase in uterine gland number and cell height with no increase in uterine weight.
At the 30th and 60th PND, BCA and E2 groups showed widely spaced lobulated glands with increase in both size and number of acini along with the presence of collapsed ducts in some instances. In the DMSO group, a consid- erable degree of vacuolation was noted in the acinar structures. These results are similar to that of De Rijk et al. (30), who revealed pseudoluminal structures formed by variably sized cells as a result of steroid application for long periods.
Mature GCD were absent in the BCA, E2, and DMSO groups indicating the involvement of striated duct cells in the changes caused by these compounds. A similar impairment was proposed by Habermann et al. (31) in the prostate following a brief exposure to estrogens prob- ably via a reduction in branching morphogenesis and the
Table 4 Tabulated means and standard deviations (SD) of Cas-3 gene expression in all groups at different ages PND Control BCA E2 DMSO P value 6 PND 1.000 ± 0.0 1.695 ± 0.03700 0.4268 ± 0.009315 0.2217 ± 0.08114 <0.0001 15 PND 1.000 ± 0.0 1.512 ± 0.5534 0.2710 ± 0.09920 1.150 ± 0.7321 <0.0001 30 PND 1.000 ± 0.0 1.244 ± 0.7914 1.386 ± 0.8780 1.150 ± 0.7321 0.8060 60 PND 1.000 ± 0.0 1.103 ± 0.09541 0.5246 ± 0.1999 0.4530 ± 0.001720 <0.0001
Table 5 Tabulated means and standard deviations (SD) of EGF gene expression in all groups at different ages following euthanization
PND Control BCA E2 DMSO P value 6 PND 1.000 ± 0.0 1.695 ± 0.7134 1.101 ± 0.4632 1.224 ± 0. 4634 0.0346 15 PND 1.000 ± 0.0 1.053 ± 0.1739 1.463 ± 0.2078 0.9913 ± 0.1421 0.0029 30 PND 1.000 ± 0.0 0.5065 ± 0.2045 0.2166 ± 0.04627 0.1885 ± 0.040 <0.0001 60 PND 1.000 ± 0.0 1.103 ± 0.09541 0.5246± 0.1999 0.4530 ± 0.00172 0.5621 Number of samples in each group, 6.
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Fig. 5 Effect of prepubertal exposure to 500 μg/g body weight (BW) of BCA, 500 ng/g BW of E2, and 500 μg/g BW of vehicle DMSO on cas-3 gene expression in the submandibular salivary gland of female rats at 6th (A), 15th (B), 30th (C), and 60th (D) PND using real-time PCR. The values represent the means ± SD (n = 6). Intergroup differences (Tukey test) are indicated by the following symbols: *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, non-significant and Cont., control.
Fig. 6 Effect of prepubertal exposure to 500 μg/g body weight (BW) of BCA, 500 ng/g BW of E2, and 500 μg/g BW of vehicle DMSO on EGF gene expression in the submandibular salivary gland of female rats at 6th (A), 15th (B), 30th (C), and 60th (D) PND using real-time PCR. The values represent the means ± SD (n = 6). Intergroup differences (Tukey test) are indicated by the following symbols: *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, non-significant and Cont., control.
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blockage of epithelial cell differentiation. Rodent submandibular glands are known to be a
major source of salivary EGF. EGF mRNA expression demonstrated a gradual increase in the control group, reaching its peak at the 60th PND. This increase coin- cided with the appearance of GCD (25,32). However, in the experimental groups, except for elevated levels of EGF at PND 15 in the E2 group, no signficant differ- ences were observed between the other groups and the controls. This may be explained by the ability of estrogen to induce the mRNA and protein expressions of both EGF and its receptor in rodent uterus. Moreover, production of EGF may be essential for estrogen-induced responses (29,30,33). The increase in EGF levels observed in the present study is not in accordance with previous studies where estrogen has been shown to inhibit EGF expres- sion, suggesting a tissue-specific dual effect (24,33-35).
Although many previous studies (reviewed in Gresik et al.) (25,36) have shown that EGF protein and mRNA levels are correlated with each other and with the number of GCDs in rodents with experimentally reduced or absent GCD, or experimentally increased GCD, the findings of the current study indicate that the molecular mechanism controlling EGF secretion is independent of that controlling GCD growth. This idea regarding independent developmental and secretory regulatory pathways has been previously suggested by Siminoski et al. (37), who proposed that submandibular NGF and EGF could be controlled by similar cellular or molecular mechanisms independent of the regulation of general- ized growth responses. Additionally, Kouidhi et al. (32) proposed that low doses of genistein and vinclozolin may have targeted GCD morphogenesis and reduced, though non-significantly, EGF and NGF mRNA levels in the submandibular salivary gland via independent pathways. A similar decrease was observed at PND 30 in the present study.
Homeostasis and sculpting of the submandibular gland are regulated by a balance between proliferative activity and deletion of cells via apoptosis and/or autophagy. Cas-3 expression was monitored, as it is one of the prin- cipal intracellular effectors of apoptosis. In the control group, the pattern of Cas-3 expression during the first two weeks was similar in both terminal transferase-mediated dUTP nick-end labeling (TUNEL) and modified in situ TUNEL techniques, which detect apoptotic cells; thus, implying apoptosis and/or autophagy in the transient structures of the gland (24,38). The continuous expres- sion of Cas-3 mRNA at the 30th and 60th PND reflects the non-apoptotic role of caspase 3 and implies its role in the differentiation of the cells since the gland is in a state
of differentiation rather than proliferation. These results agree with the findings reported in
previous studies where caspases have been revealed as dynamic regulatory molecules during cell differentiation (in neural cells and salivary gland in drosophila), cell morphology, stem-cell maintenance, tissue regeneration and actin-cytoskeleton reorganization (38-41). On the contrary, Kouidhi et al. (32) demonstrated the absence of caspase 3 at PNDs 21 and 35 in their study.
The BCA group showed a significant increase in Cas-3 expresson in both the 6th and 15th PND. On the other hand, Cas-3 expression in the DMSO group was similar to that seen in the studies by Galvao et al. (42) and Yuan et al. (43); they revealed non-caspase-3 activation, decreased cell viability, mitochondrial distortion, and membrane potential impairment in astrocyte cell cultures after exposure to DMSO. The ability of DMSO to cause membrane loosening, pore formation, and bilayer collapse may be responsible for the structure distortion (44).
Cas-3 expression levels in the E2 group were lower than that of the control group, which may be due to the anti-apoptotic property of estrogen leading to the inhibition of cell apoptosis in the submandibular gland. Nevertheless, Tsinti et al. (18) reported the absence of an impact of estrogen on cell apoptosis in vitro via salivary gland cell culture. These differences in observation may be attributed to the more complicated in vivo environment when compared with the in vitro environment (29,45,46).
The presence of ERs is an important factor for cells to respond to hormones; however, estrogens can act upon the cell via independent pathways (47). Sakabe et al. (48) demonstrated the presence of ERs in excretory ducts but not in acini, in relation to EGF production. Despite the controversy regarding the presence of ERs in salivary glands, the present study did not only confirm the presence of ERβ receptors in SMG, but also shed light on its role during differentiation. The expression was cytoplasmic in the early stages of development, and subsequently shifted to a nuclear expression. This is in agreement with the results reported by Mishra et al. (14), where ERα did not appear during the proliferating phase but appeared during differentiation in the mammary gland.
Conflicting patterns of expression were observed in the E2 and BCA groups when compared to controls at the 60th PND; the number of cells with positive nuclei were low in the E2 group and high in the BCA group. These findings are consistent with those reported by Patisaul et al. (49), wherein, ERβ was down-regulated by estrogen in a region specific manner in the rat brain, whereas exposure to coumestrol (phytoestrogen) is
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thought to modulate ERβ. Chen et al. (50) observed the same contrasting pattern between E2 and a group of phytoestrogens (genistein, resveratrol, and quercetin) in the breast cancer cell line MCF-7.
E2 is known, at least in part, to induce cancerous trans- formations by causing deleterious mutations through the formation of reactive oxygen species. Oxidative stress inactivates ERβ by inhibiting the receptor’s dimeriza- tion through alteration of the second zinc-finger motif in the ERβ structure, and therefore, destabilizing its DNA-binding capacity (51). As a result, ERβ loses its ability to regulate various genes. Such mechanism may be responsible for the decrease in ERβ expression and associated damage.
Acknowledgments We would like to thank our supervisors and members of the Oral Biology Department for their help in this work.
Conflict of interest The authors have no conflict of interest to declare.
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