Cheek Pouch Epithelium Research Articles

Evaluation of differential white blood cell count and cheek pouch epithelium in 7,12-dimethylbenza[a]anthracene hamster carcinogenesis model, managed with three phytochemicals

Evaluation of differential white blood cell count and cheek pouch epithelium in 7,12-dimethylbenza[a]anthracene hamster carcinogenesis model, managed with three phytochemicals

Evaluation of differential white blood cell count and cheek pouch epithelium in 7,12-dimethylbenza[a]anthracene hamster carcinogenesis model, managed with three phytochemicals

Objectives: Nigella sativa (NS), thymoquinone (TQ), and epigallocatechin-3-gallate (EGCG) are phytochemicals that might have antioxidant protective potentials on the hamster cheek pouch epithelium (HCPE). We aimed at evaluating and comparing the potential therapeutic outcomes of these 3 phytochemicals by analysis of peripheral white blood cells (WBCs) counts. Materials and Methods: NS whole oil, TQ and EGCG were administered before, with or after 7,12-dimethylbenza[a]anthracene (DMBA) painting the hamster left cheek pouch. Before sacrificing each animal, 2 ml of blood was withdrawn into a fine heparin-containing tube to estimate the total WBCs, lymphocytes, MID cells, and granulocytes counts by an automatic count system. All cheek pouches were surgically excised and examined with light microscope. Results: Severe epithelial dysplasia was evident after 6 weeks of DMBA administration, and when NS was given for 2 weeks followed by DMBA for 6 weeks. When NS or EGCG were given for 2 weeks then continued with DMBA for 6 weeks, mild dysplasia was seen. When DMBA was given for 6 weeks followed by NS or TQ for 6 weeks, mild dysplasia was noted. Administration of DMBA for 6 weeks resulted in significant reduction in total WBCs and lymphocytes counts compared to healthy controls. Administration of NS or TQ for 2 weeks resulted in significant elevation in lymphocytes count compared to healthy controls. Significant elevation in total WBCS and lymphocytes counts was noted when EGCG was given for 2 weeks and continued with DMBA for other 6 weeks. Similar results were noted when DMBA was given for 6 weeks followed by TQ for 6 weeks when compared to NS, DMBA or healthy controls. Discussion: The three phytochemicals showed different levels of protection against DMBA carcinogenic activity, more specifically, TQ and NS had higher therapeutic potential and might be used for treatment and/or preventive management of oral cancer in the future. Conclusion: However, further investigations are required to address the mechanism of action and feasibility of clinical application of each phytochemical.

EVALUATION OF DIFFERENTIAL MONOCYTES IN THE DMBA/HCPE CARCINOGENESIS MODEL UNDER THE INFLUENCE OF SOME PHYTOCHEMICAL AGENTS

Dimethylbenza-a-anthracene (DMBA), is a potent carcinogen that has an immunosuppressive effect on hamsters. Nigella sativa whole oil (NSO) and thymoquinone (TQ), are phytochemicals that have an antioxidant and protective action on the hamster cheek pouch epithelium (HCPE/DMBA) carcinogenesis model. The aim is to find which of the two ingredients TQ & NSO has more immune-enhancing activity comparable with its protective and/or chemotherapeutic activity against the carcinogenic effect of DMBA on HCPE, through evaluation of different peripheral monocytes. Material &methods: NS whole oil (NSO), and thymoquinone (TQ) were given before-, with- or after DMBA painting the hamster’s left cheek pouch. Before each animal’s sacrificing, two ml of blood was drawn in a fine heparinized capillary tube to estimate the total WBCs, total lymphocytes, MID cells, and granulocyte’s count by an automatic count system (Cell-DYN1700).Blood results were statistically analyzed using One-way ANOVA test between groups for each phase followed by LSD (Least Square Difference) test for multiple comparisons between different phases. All pouches were surgically-excised and examined microscopically to compare the histopathologic changes of DMBA-painted pouches, NSO-and TQ-treated groups, with untreated pouches as well as pouches of the untreated group.Histopathologic results: the groups given each of the tested ingredients for 2 wks showed normal appearing thin epithelium, with slight hyper-keratinization as compared with untreated pouches. Severe epithelial dysplasia was seen after 6 wks of DMBA, but when NSO or TQ was given for 2wks followed by DMBA for 6wks, mild dysplasia was seen i.e. both have protective effect. When DMBA for 6wks was followed by NSO or TQ for 6wks mild dysplasia was recorded .i.e. both had a therapeutic effect through regressing severe dysplasia to mild dysplasia in 6 weeks. Mononuclear cells results: DMBA for 6wks resulted in statistically significant reduction in total WBCs &lymphocyte counts than control group, as well as when DMBA for 6wks was followed

Ex vivooptical metabolic measurements from cultured tissue reflectin vivotissue status

Optical measurements of metabolism are ideally acquired in vivo; however, intravital measurements are often impractical. Accurate ex vivo assessments would greatly broaden the applicability of optical measurements of metabolism. We investigate the use of live tissue culture experiments to serve as a surrogate for in vivo metabolic measurements. To validate this approach, NADH and FAD fluorescence intensity and lifetime images were acquired with a two-photon microscope from hamster cheek pouch epithelia in vivo, from biopsies maintained in live tissue culture up to 48 h, and from flash-frozen and thawed biopsies. We found that the optical redox ratio (fluorescence intensity of NADH/FAD) of the cultured biopsy was statistically identical to the in vivo measurement until 24 h, while the redox ratio of the frozen-thawed samples decreased by 15% (p<0.01). The NADH mean fluorescence lifetime (τm) remained unchanged (p>0.05) during the first 8 h of tissue culture, while the NADH τm of frozen-thawed samples increased by 13% (p<0.001). Cellular morphology did not significantly change between in vivo, cultured, and frozen-thawed tissues (p>0.05). All results were consistent across multiple depth layers in this stratified squamous epithelial tissue. Histological markers for proliferation and apoptosis also confirm the viability of tissues maintained in culture. This study suggests that short-term ex vivo tissue culture may be more appropriate than frozen-thawed tissue for optical metabolic and morphologic measurements that approximate in vivo status.

Parallel frequency-domain optical coherence tomography scatter-mode imaging of the hamster cheek pouch using a thermal light source

We use a parallel frequency-domain optical coherence tomography (FDOCT) system to generate a scatter-mode image of the hamster cheek pouch epithelium. To our knowledge, this is the first optical coherence tomography (OCT) image of a biological sample obtained using a thermal light source in the frequency domain. The system employs an imaging spectrometer to acquire depth-resolved profiles from adjacent spatial points without the need for any scanning. To enable this imaging modality, we have considered that signals originating from multiple depths combine in a different manner in FDOCT compared to time-domain optical coherence tomography (TDOCT). Because a multicomponent FDOCT signal is a coherent sum, it is necessary to limit the number of modes that contribute to the detected signal. Conversely, multicomponent TDOCT signals can be represented as incoherent sums, where increasing the number of modes improves the signal.

Effect of Eriobotrya japonica Seed Extract on 5-Fluorouracil-Induced Mucositis in Hamsters

In this study, we investigated the effects of an Eriobotrya japonica seed extract (ESE) on mucositis using a 5-fluorouracil (5-FU)-induced mucositis hamster model. This model was prepared by intraperitoneally administering 90 mg/kg of 5-FU to hamsters on Day 1, scratching 1 cm(2) of the left cheek pouch of hamsters with a wire brush on Days 2, 3, and 4, and intraperitoneally administering 60 mg/kg of 5-FU on Day 5. Mucositis was evaluated based on the mucositis score at the mucositis site, left cheek pouch thickness, histological findings on HE staining, and plasma lipid peroxide levels. On Day 10, the mucositis score and left cheek pouch thickness in the ESE group were significantly lower than those in the tap water group. Histologically, the two groups showed a defect of the cheek pouch epithelium on Day 6. On Day 10, epithelial injury and bacterial infection were noted in the tap water group. However, in the ESE group, similar findings were not observed. On Day 6, the plasma lipid peroxide level in the tap water group was significantly higher than that in the normal group. In the ESE group, the plasma lipid peroxide level was significantly lower than that in the tap water group. These results suggest that ESE is useful for treating chemotherapy-induced mucositis.

Smokeless tobacco‐induced lamellar body abnormalities

To compare the morphological changes and quantitative distribution of lamellar bodies (Lb) (membrane coating granules) in the hamster cheek pouch epithelium with smokeless tobacco (ST). Archives of experimental material from previously published studies [S. Ashrafi, A. Das, R. Worawongvasu, B. Mehdinejad and J. Waterhouse (1992) Scanning Microscopy6: 183] were utilized. Animals in experimental group received most ST (snuff) in their right pouch, 5 days weekly, for 24 months, while no snuff was given to control group. After 24 months, the epithelial tissues were processed for electron microscopic study. Volume densities of Lb were assessed by morphometry. Densities of Lb in the two groups, experimental vs control. In the control, Lb extruded their contents into the intercellular spaces of upper granular layers and in between the last granular cell layers and keratin layers to form a permeability barrier. Conversely, in the smokeless tobacco-treated epithelium, the majority of the Lb that were formed remained inside and accumulated within the granular cells, without extruding their contents into the intercellular spaces to form a lipid compound permeability barrier. Commercial alkaline ST may have contributed to the abnormal accumulation of Lb in the granular cell layer and affected the extrusion process of Lb to form an incomplete permeability barrier in the oral epithelium.

Expression of bone sialoprotein in mineralized tissues of tooth and bone and in buccal-pouch carcinomas of Syrian golden hamsters

The expression of bone sialoprotein (BSP) is normally restricted to mineralized connective tissues of bones and teeth where it has been associated with mineral crystal formation. However, recent studies have revealed ectopic expression of BSP in various lesions, including oral and extraoral carcinomas, in which it has been associated with the formation of microcrystalline deposits and the metastasis of cancer cells to bone. To develop a model to study the induction of BSP in carcinoma development, BSP expression in squamous-cell carcinomas induced by chemical carcinogen in the hamster cheek-pouch epithelium was investigated. Hamster BSP cDNA was first isolated and characterized, then used to prepare probes for Northern and in situ hybridization. The protein sequence of hamster BSP displayed 86% amino acid identity with a consensus mammalian BSP sequence and retained polyglutamate sequences, the RGD sequence and sites of phosphorylation, glycosylation and sulphation. The tissue-specific expression of hamster BSP mRNA and protein was confirmed by in situ hybridization and immunolocalization in developing tissues. Squamous-cell carcinomas induced in the buccal pouches of 5-week-old male Syrian golden hamsters treated with chemical carcinogen had BSP mRNA and BSP in the proliferating neoplastic epithelium. In contrast, neither BSP mRNA nor the protein could be detected in the stroma within which islands of the transformed tissue had formed. Thus, the hamster cheek pouch is a well-characterized model that can be used to study the induced expression of BSP in association with the development of squamous-cell carcinomas.

Cytochrome P-450 omega-hydroxylase senses O2 in hamster muscle, but not cheek pouch epithelium, microcirculation.

The goal of this study was to investigate the role of cytochrome P-450 omega-hydroxylase in mediating O2-induced constriction of arterioles in the microcirculation of the hamster. Male Golden hamsters were anesthetized with pentobarbital sodium, and the cremaster muscle or cheek pouch was prepared for observation by intravital microscopy. Arteriolar diameters were measured during elevations of superfusate PO2 from approximately 5 to 150 mmHg. Arteriolar responses to elevated PO2 were determined in the cremaster muscle, in the retractor muscle where it inserts on the cheek pouch, and in the epithelial portion of the cheek pouch. Elevation of superfusion solution PO2 caused a vigorous constriction of arterioles in the cremaster and retractor muscles and in the epithelial portion of the cheek pouch. Superfusion with 10 microM 17-octadecynoic acid, a suicide substrate inhibitor of cytochrome P-450 omega-hydroxylase, and intravenous infusion of N-methylsulfonyl-12,12-dibromododec-11-enamide, a mechanistically different and highly selective inhibitor of cytochrome P-450 omega-hydroxylase, caused a significant reduction in the magnitude of O2-induced constriction of arterioles in the cremaster and retractor muscles. However, arteriolar constriction in response to elevated PO2 was unaffected by 17-octadecynoic acid or N-methylsulfonyl-12,12-dibromododec-11-enamide in the epithelial portion of the cheek pouch. These data confirm that there are regional differences in the mechanism of action of O2 on the microcirculation and indicate that cytochrome P-450 omega-hydroxylase senses O2 in the microcirculation of hamster skeletal muscle, but not in the cheek pouch epithelium.

Differential expression of type I cytokeratins in hamster cheek pouch epithelium following treatment with dimethylbenzanthracene.

Cytokeratin (CK) expression in untreated, paraffin-treated or dimethylbenzanthracene (DMBA)-treated hamster cheek pouch epithelium was investigated utilizing monoclonal antibodies AE1 or AE3, which react with type I or type II CKs, respectively, and by in situ hybridization utilizing type I CK-specific probes. The latter were isolated from a cDNA library of hamster cheek pouch mRNA and designated CK 13 and CK 10 based on their respective homologies (> 95% amino acids) with murine CK 13 and human CK 10. Treatment of hamster cheek pouch epithelium with DMBA resulted in increased expression of type I CK, detected immunohistochemically with monoclonal AE1, but decreased expression of type II CKs detected with AE3. Despite an overall increase in type I CKs, in situ hybridization demonstrated differential expression of type I CKs with altered distribution of CK 13 mRNA and reduced expression of CK 10 mRNA, providing additional sensitive markers for DMBA-associated changes in CKs. These changes were constant at 2 to 22 weeks in the pre-neoplastic and neoplastic epithelium following the initial application of DMBA.

Lipids of hamster cheek pouch epithelium.

The hamster cheek pouch is a much used but incompletely understood experimental model. In particular, the cheek pouch epithelial lipids, which are important for permeability barrier function as well as other aspects of epithelial biology, have not been completely characterized. In the present study, the complete lipid class composition has been determined by thin-layer chromatography in conjunction with photodensitometry. The major lipid classes were phospholipids, free sterols, and ceramides. Minor amounts of monohexosylceramides, sterol esters, fatty acids, and triglycerides were also present. Significant amounts of covalently bound omega-hydroxyceramide was also detected. Transmission electron micrographs reveal extensive, largely paired, lipid bilayers in the intercellular spaces of the stratum corneum.

Triclosan inhibits sodium lauryl sulphate-induced changes in expression of cytokeratin genes in hamster cheek pouch epithelium.

Triclosan is known to reduce the untoward side effects of sodium lauryl sulphate (SLS). The aim of the present study was to determine whether triclosan can inhibit SLS-induced changes in expression of cytokeratin (CK) genes in hamster cheek pouch epithelium. With a hybridohistochemical technique, using specific human cRNA probes, hamster CK mRNAs were identified by immunological detection of heterologous hybrids. In contrast to application of SLS-containing paste, application of paste containing SLS together with triclosan produced no marked changes in expression and distribution patterns of CK mRNAs, compared to the normal cheek pouch epithelium. Therefore, we may accept that triclosan inhibits the effect of SLS on CK gene expression. However, the mechanism of this protection remains elusive. Conversely, the epithelial hyperplasia induced by application of SLS was histologically identical to that induced by application of SLS and triclosan. This suggests that the changes in CK gene expression identified in the present study are not a simple consequence of epithelial hyperplasia, but rather are specific to the irritating agent. Establishment of the fact that SLS may influence gene expression, and that this may be prevented by triclosan, may be helpful in research on the cytological effects of SLS and the elucidation of protection mechanisms of triclosan against side effects of SLS.

The effect of sodium lauryl sulphate and triclosan on hamster cheek pouch mucosa.

It has recently been shown that triclosan protects the human skin from the inflammation that may be caused by exposure to sodium lauryl sulphate (SLS). The aim of the present study was to examine whether triclosan can protect the hamster cheek pouch mucosa from the irritation caused by exposure to SLS. After four daily applications of a paste containing SLS, the epithelium of the hamster cheek pouch showed consistently prominent structural changes, especially basal hyperplasia, acanthosis, hypergranulosis, and hyperkeratosis. Identical morphological changes were also observed after applications of a paste containing SLS together with triclosan. In contrast, after applications of a paste containing triclosan alone, the cheek pouch mucosa revealed a histological structure essentially similar to the non-treated control mucosa. From these results, we may conclude that SLS, but not triclosan, irritates the hamster cheek pouch epithelium. Moreover, triclosan does not protect the cheek pouch mucosa against structural changes induced by SLS. It must be taken into account that triclosan does not always offer protection against the side-effects of SLS.

Circadian variation of DNA replication in hamster cheek pouch epithelium analysed by tritiated thymidine labelling, flow sorting and autoradiography: no resting S phase cells in the normal epithelium

Circadian variation of DNA replication in hamster cheek pouch epithelium analysed by tritiated thymidine labelling, flow sorting and autoradiography: no resting S phase cells in the normal epithelium

Circadian variation of DNA replication in hamster cheek pouch epithelium analysed by tritiated thymidine labelling, flow sorting and autoradiography: no resting S phase cells in the normal epithelium

In the normal hamster cheek pouch epithelium, cell proliferation takes place with a pronounced circadian rhythm. We tested our previous hypothesis that all cells having S phase DNA content are actively synthesizing DNA and thus participating in the daily cohort of proliferating cells. We found no evidence of resting S phase cells in the normal epithelium. Using labelling with tritiated thymidine followed by fluorescence activated cell sorting according to DNA content and by autoradiography of the sorted nuclei, it was demonstrated that during the 24 h period almost all cells with mid S phase DNA content were active in DNA synthesis.

Differential expression of cytokeratin mRNA in hamster squamous epithelia as observed by in situ hybridization using human cytokeratin cRNA probes.

In this hybridohistochemical study, we investigated the expression of cytokeratin (CK) mRNA in the keratinizing squamous epithelium of hamster cheek pouch and esophagus, using eight different digoxigenin-labelled RNA probes complementary to human CK mRNAs. CK 4, CK 6, CK 8, CK 14, and CK 15 RNA probe obviously hybridized with hamster counterpart mRNA(s) in the cheek pouch as well as in the esophageal epithelium. However, using the CK 10-specific RNA probe, only the cheek pouch epithelium exhibited a positive reaction. We were not able to detect any positive signal for the CK 18 or for the CK 19 RNA probe. We observed three different CK mRNA distribution patterns in the cheek pouch epithelium, and four in the esophageal epithelium. The differences in expression and distribution pattern of CK mRNAs between the two types of epithelia suggest that the hamster CK polypeptide family comprises at least six different species. We also conclude that human cRNA probes for CK mRNAs may provide a way to detect changes in CK expression and distribution during induced non-neoplastic and neoplastic changes in the hamster cheek pouch model. This may also help to elucidate the molecular pathogenesis of squamous-cell carcinoma.

Effects of nicotine and tobacco-specific nitrosamines on hamster cheek pouch and gastric mucosa

Fifty-two hamsters were divided into 6 groups and their cheek pouches treated with either 0.01% NNN, 0.01% NNN and 6% nicotine, 0.01% NNK, 0.01% NNK and 6% nicotine, 6% nicotine, or sesame oil. After painting each pouch 3 times per week for 13 months, the animals were killed and specimens from the cheek pouch and forestomach examined. Cheek pouch epithelium showed more frequent histologic changes, including hyperplasia, hyperkeratosis and, in one animal, moderate dysplasia, when nicotine was combined with NNN than after treatment with NNN (or nicotine) alone. There was a higher frequency of hyperplasia with hyperkeratosis in the forestomach when nicotine was combined with NNK than following NNK, or nicotine treatment alone, and squamous cell papillomas were evident in animals treated with both NNK and nicotine. These results suggest that in mucosal tissues nicotine may enhance the effect of weak carcinogens such as the nitrosamines.

Iron deficiency reduces cytochrome concentrations of mitochondria isolated from hamster cheek pouch epithelium

The effects of iron deficiency on the aerobic pathway of energy metabolism were studied using mitochondria isolated from epithelial cells from the hamster cheek pouch. A statistically significant reduction in the concentrations of cytochromes aa3, b and c (P less than 0.05), a reduction (P = 0.064) in cytochrome cl and altered cytochrome ratios were found in the mitochondria of iron deficient compared to normal animals. State 4 respiration was demonstrated in the mitochondria of both normal and iron deficient animals but state 3 respiration could not be demonstrated; this suggests uncoupling of oxidative phosphorylation which may be an artefact associated with the separation of epithelium from its connective tissue. Nevertheless we conclude that the reduction in cytochrome concentration is a real effect of iron deficiency which may explain, at least in part, the reduction of both energy production and cell proliferation seen in oral epithelia under these conditions.

Cytokeratins in hamster cheek pouch epithelium during DMBA‐induced carcinogenesis

The pattern of keratin expression in hamster cheek pouch epithelium during 15-wk of DMBA-induced carcinogenesis was studied. The sequential changes in cytokeratins of premalignant and malignant tissues and comparative investigation of normal epithelial tissues were examined during a weekly sequential DMBA-induced chemical carcinogenesis. Keratin polypeptides of normal pouch epithelium appear in a molecular weight range of 43-67 kd and 5-6 proteins can be identified. The disappearance of high molecular weight keratin (61-67 kd) was observed from the 6-wk DMBA-treated premalignant group to the 15-wk DMBA-treated malignant group. An additional keratin polypeptide was noted initially on the 11th-wk-DMBA-treated group and remained to the 15th-wk-DMBA treated group.

In vivo effects of Sn-1,2-dioctanoylglycerol, TPA and A23187 on hamster cheek pouch epithelium.

Cheek pouches of male Syrian golden hamsters were topically treated with a single dose of TPA (.5 microgram), calcium ionophore A23187 (75 micrograms) or Sn-1,2-dioctanoylglycerol (DiC8) (500 micrograms) dissolved in 0.25 ml acetone. Acetone-treated animals served as controls. After 48 h the mitotic index for the control group was 1.1 +/- 0.1 per 1 mm of the basement membrane length. All the test congeners exhibited higher mitotic indices than controls: TPA (4.8 +/- 0.4), A23187 (3.9 +/- 0.3), DiC8 (2.1 +/- 0.2). All groups exhibited an increase in the epithelial thickness manifested by cellular hyperplasia. The treatment of the pouches with the anti-inflammatory agent fluocinolone acetonide inhibited the mitogenic and hyperplasiogenic affects on the epithelium induced by the various test chemicals. These studies indicate a possible role of calcium-phospholipid dependent protein kinase (protein kinase C) in the mediation of oral epithelial cell proliferation.