Cytogenetic alterations in buccal mucosa cells of chewers of areca nut and tobacco

Objective

The rationale of the study was to evaluate the cytological alterations especially micronucleus (MN) and other nuclear anomalies in buccal mucosa cells of chewers to understand the genotoxic and clastogenic potential of chewing mixture (containing areca nut and tobacco as main ingredients).

Methods

The buccal cytome assay involves the examination of epithelial smear to determine micronucleated cell and other nuclear anomalies after the Feulgen plus light green staining. The assay was applied to exfoliated buccal mucosa cells of 262 subjects [non-chewers – 161 and chewers – 101 (includes 20 subjects with OSMF)] and 1000 cells per individual were examined microscopically. Nuclear anomalies were compared among chewers, non-chewers and OSMF subjects and correlated with consumption of quids per day and duration of chewing in years.

Results

MN cells were found significantly (p < 0.0001) higher among chewers and OSMF subjects as compared to non-chewers. Further analysis indicated that MN was significantly higher in OSMF subjects with respect to even chewers. Nuclear buds were significantly higher (p < 0.0001) in OSMF subjects as compared to chewers as well as non-chewers. Nuclear anomalies viz. binucleated, karyorrhexis and karyolysis were also considerably higher in OSMF subjects as compared to non-chewers.

Conclusion

The MN and other nuclear anomalies reflected genetic damage and cytotoxicity, associated with tobacco and areca nut consumption. Further, these data reveal a risk for development of OSMF among chewers of mixture containing areca nut and/or tobacco, as all the OSMF subjects were chewers.     

A light microscopic study of fibrosis involving muscle in oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a common pre-malignant condition of the mouth in arecanut chewers in Indian subcontinent. It is characterized by restriction in opening of the mouth, tongue protrusion, and cheek flexibility. Reports of muscle changes in OSF, using light microscopy are few. Incisional biopsy sections of thirty OSF patients were stained with Masson's Trichrome stain, to study the fibrosis involving the muscle. Wide spectrum of changes were observed where fibrosis was being evident sub-epithelially, not extending in to the muscle to cases where only a few remnants of muscle fibers were seen and the missing muscle bundle area being replaced by fibrous tissue. The probable mechanism by which these changes occur is discussed.     

Epidemiological survey of oral submucous fibrosis in Xiangtan City, Hunan Province, China

Abstract Oral submucous fibrosis is a high risk precancerous condition and is suggested to be caused by areca nut chewing. Areca nut chewing is popular in Hunan Province of China, and is more concentrated in Xiangtan City. Two and nine cases of oral submucous fibrosis (OSF) were first noticed in 1984 and 1985 respectively, and an epidemiologic survey was subsequently performed in 1986. The epidemiologic method of cluster sampling was used. The Yuhu District, one of the five urban districts of the Xiangtan City with a population of 100 000 was selected as a whole body in the survey. 57 independent units of various professions were randomly selected as group samples and more than 70% of subjects in each unit were examined. Definite fibrous band on palpation was used as a main diagnostic criterion for OSF. A total of 11 046 individuals were examined; among them were 3 907 areca nut chewers (35.37%) and 7 139 non-chewers (64.63%). 335 cases of OSF were found, comprising a prevalence rate of 3.03%. The disease involved mainly the middle third of the oral cavity. All of the OSF cases were areca nut chewers. No case had been found in non-chewers. Four cases of oral carcinoma were found on the basis of OSF, the malignant transformation rate was 1.19%. The high prevalence of OSF may be due to areca nut chewing plus extensive and heavy use of hot pepper in Xiangtan people. The result supports the role of the areca nut as an etiologic factor in the development of OSF. The low malignant transformation rate of 1.19% compared with the 7.6% in an Indian report may be because Xiangtan people chew areca nut without tobacco.     

兜甲蛋白和细胞色素P4503A5在口腔黏膜下纤维化中的表达及意义

目的探讨兜甲蛋白（LOR）和细胞色素P450 3A5（CYP 3A5）在口腔黏膜下纤维化颊黏膜（OSF）和正常颊黏膜中的表达以及在上皮防御作用中的意义。方法首先采用免疫组化染色检测66例OSF颊黏膜和14例正常颊黏膜中LOR和CYP 3A5这2种蛋白的表达和定位,然后采用Western blot和RT-PCR方法分别在OSF患者颊黏膜和正常人颊黏膜中检测LOR和CYP 3A5的蛋白和mRNA表达。结果42例（63.6%）OSF呈LOR阳性表达,其在早、中期之间差异有统计学意义（P<0.05）,而中、晚期之间差异无统计学意义（P＞0.05）;正常颊黏膜中CYP 3A5均呈阳性表达,5例（7.6%）OSF出现棘细胞弱阳性表达,33例（50%）出现内皮细胞弱阳性表达,与病理分期呈负相关（P<0.05）。RTPCR显示LOR和CYP 3A5 mRNA在OSF与正常黏膜中表达的差异均有统计学意义（P<0.05）;而Western blot检测显示,只有CYP 3A5蛋白表达在两者间差异有统计学意义（P<0.05）。结论LOR和CYP 3A5的异常表达是OSF黏膜上皮防御能力改变的表现,它们在OSF发生发展和癌变过程中可能发挥了重要作用。     

口腔粘膜下纤维化、白斑及鳞癌上皮细胞内皮素1表达的定量分析

目的 :探讨内皮素 (ET 1)在口腔癌变发病机制中的作用和意义。方法 :采用免疫组化染色SABC法和图象分析技术 ,对人口腔粘膜下纤维化 (OSF) 10例、白斑 (OLK) 9例、鳞状细胞癌 (SCC) 14例及正常口腔粘膜(NOR) 10例的上皮细胞ET 1表达进行定量分析。结果 :①ET 1在OSF、OLK、SCC组织中的表达增强 ,阳性颗粒主要位于上皮棘细胞、基底细胞的胞浆胞膜上。ET 1表达阳性率和含量显著高于正常对照 (P <0 .0 1)。②OLK、SCC上皮细胞ET 1含量呈显著增加趋势 (P <0 .0 5 )。③OSF上皮细胞ET 1含量显著高于OLK(P <0 .0 5 ) ,与SCC相比无显著性差异 (P >0 .0 5 )。结论 :ET 1含量在口腔癌前病变至癌变过程中可能存在一种量变关系 ,OSF中ET 1过量表达可能提示其上皮细胞的癌变潜能 ,ET 1与OSF、OLK、SCC的发生发展密切相关     

口腔黏膜下纤维性变中MMP-2基因的表达及意义

目的：检测口腔黏膜下纤维性变中基质金属蛋白酶-2（MMP-2）的表达并探讨其病理意义.方法：抽提11例口腔黏膜下纤维化组织和10例正常口腔黏膜组织的总RNA,通过逆转录聚合酶链反应（PF-PCR）检测MMP-2mRNA在口腔黏膜下纤维性变患者颊黏膜中的表达并与正常口腔黏膜进行比较.结果：口腔黏膜下纤维性变患者颊黏膜组织中MMP-2 mRNA表达高于正常颊黏膜（p＜0.05）.结论：MMP-2基因的表达与口腔黏膜下纤维性变组织重塑过程密切相关.     

口腔粘膜下纤维化中ET-1mRNA表达的研究

目的:通过研究内皮素-1(endothelin-1,ET-1)在口腔粘膜下纤维化中mRNA的表达,探讨ET-1与OSF(oral submucous fibrosis)病变发生发展的关系。方法:选取30例OSF患者,其中早、中、晚期组各10例,5例健康志愿者作为对照组,每例研究对象取其口腔颊粘膜,运用逆转录聚合酶链反应(RT-PCR)研究ET-1在OSF中mRNA的表达水平。结果:ET-1的mRNA在OSF各期中的表达均较正常口腔粘膜高(P<0.05);ET-1mRNA在OSF早期的表达均明显低于中、晚期(P<0.05),其中期的表达与晚期相比差异无显著性(P>0.05)。结论:各种调节因素在基因转录水平对ET-1表达的上调以及由此造成的ET-1基因的异常表达可能与OSF病变的发生发展密切相关。     

口腔黏膜下纤维化中VEGF mRNA的表达研究

目的:研究口腔黏膜下纤维化(oral submucous fibrosis,OSF)患者早、中、晚期组中血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA的表达,探讨VEGF与OSF微血管病变发生发展的关系.方法:选取OSF患者30例,其中早、中、晚期组各10例,5例健康志愿者为对照组.每例研究对象取其1:3腔黏膜,运用逆转录聚合酶链反应(RT-PCR)方法,研究OSF患者早、中、晚期组中VEGF mRNA的表达水平.结果:VEGF mRNA在OSF早、中期的表达均较正常口腔黏膜高(P＜0.05):VEGF mRNA在OSF早期的表达均明显高于中、晚期(P＜0.05),其中中期的表达与晚期相比差异无显著性(P＞0.05).结论:VEGF对OSF微血管病变的影响主要在黏膜下层,各种调控因素在基因转录水平导致VEGF的高表达可能是OSF局部组织缺血的代偿.     

bFGF在口腔黏膜下纤维性变组织中的表达研究

目的:检测碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)在正常颊黏膜(normal buccalmucosa,NBM)、口腔黏膜下纤维性变(oral submucous fibrosis,OSF)上皮组织中的表达,探讨bFGF在OSF发生发展中可能发生的作用。方法:收集10例NBM、30例颊部OSF上皮组织,利用免疫组化(immunohistochemistry,IHC)、聚合酶链式反应(PCR)和Western Blotting(WB)的方法研究bFGF在其中的分布和表达情况。结果:IHC提示:bFGF在NBM和OSF上皮组织中均有表达。在正常黏膜中,bFGF主要表达在黏膜下层的成纤维细胞胞浆中。在OSF早期,bFGF主要表达在上皮细胞间隙,基底膜和细胞浆中,发展到中晚期后则逐渐扩大表达,广范分布在细胞核中。bFGF的阳性表达率与其病理级别呈正相关。PCR提示正常黏膜组织中有bFGF mRNA的表达。在OSF组织中,bFGF mRNA随病变的加重而表达增加。WB提示:NBM上皮组织中未探及bFGF蛋白的表达,在OSF标本中bFGF随病变的加重而表达增强。结论:bFGF在OSF的发生发展中起着一定的作用。     

Raised tissue copper levels in oral submucous fibrosis

Oral submucous fibrosis (OSF) is a well-recognised-potentially malignant condition of the oral cavity associated with areca nut chewing. Areca nut has been shown to have a high copper content compared to other commonly eaten nuts, and chewing areca nut for 5-30 min significantly increases soluble copper in whole mouth fluids. Our aims were to determine if tissue and serum concentrations of copper were raised in patients with OSF as a result of chewing areca nut. A panel of buccal mucosal biopsies from patients with OSF from Nagpur, India, was used to measure the tissue concentrations of copper by mass absorption spectrometry (MAS). By MAS, the mean tissue copper level was 5.5+/-2.9 microg/g in the OSF specimens (n=11) compared with 4+/-1.9 microg/g in the non-areca chewing controls (n=7) (P=0.2). Energy dispersive x-ray microanalysis (EDX) was used to identify the presence and distribution of the metal element. EDX showed distinct peaks corresponding to copper (Kalpha 8.04 keV; Kbeta, 8.91 keV) in the epithelium (21/23) and in the connective tissue (17/23) of the OSF specimens compared to spectra obtained from control oral biopsies from non-areca chewing subjects (n=7). These findings were confirmed by secondary ion mass spectrometry (SIMS) analysis in a small number of samples. Serum copper (17.23+/-1.80 pmol/l), caeruloplasmin (0.32+/-0.04 g/l) levels and urinary copper (0.52+/-0.26 micromol/l) in OSF patients (n=14) were within the laboratory reference ranges. The finding of copper in OSF tissue supports the hypothesis of copper as an initiating factor in OSF, playing a role in stimulating fibrogenesis by the upregulation of lysyl oxidase activity.     

口腔黏膜下纤维性变中u-PA和PAI-1的表达及意义

目的:了解尿激酶型纤溶酶原激活物(u-PA)及其抑制剂(PAI-1)在口腔黏膜下纤维性变(OSF)组织中的表达及其意义。方法:采用免疫组化染色法和图像分析法,对OSF早、中、晚期各10例及10例正常口腔黏膜组织中的u-PA及PAI-1的表达及分布进行分析。结果:OSF病变组织中u-PA和PAI-1有异常表达,其阳性物质主要分布于固有层血管内皮细胞、成纤维细胞及炎症细胞的胞质,PAI-1各期阳性表达强度均高于正常口腔黏膜组(P<0.05),u-PA早期阳性表达显著升高(P<0.05),中晚期则逐渐降低(与正常组比P>0.05)。结论:u-PA、PAI-1在OSF病变组织中的异常表达,可能是影响OSF发生发展的重要因素。     

Effects of arecoline, safrole, and nicotine on collagen phagocytosis by human buccal mucosal fibroblasts as a possible mechanism for oral submucous fibrosis in Taiwan

BACKGROUND: Oral submucous fibrosis (OSF) is associated with the betel quid chewing habit, and 86% of betel quid chewers in Taiwan are also smokers. Arecoline and safrole are major principles in the composition of betel quid, and nicotine is the main toxic ingredient of cigarettes. METHODS: To explore the pathogenesis of OSF, flow cytometry was used to compare collagen phagocytosis by fibroblasts from the normal and the OSF region of the same 15 OSF patients. RESULTS: The results indicated that heterogeneity of fibroblasts existed because collagen phagocytosis by fibroblasts from the normal region was higher than from the OSF region in the same patient. The percentage of phagocytic cells was significantly inhibited by 10, 25 and 50 microg/ml arecoline, safrole and nicotine in normal fibroblast cultures, respectively, and the percentage of phagocytic cells was significantly reduced by 25, 25 and 50 microg/ml arecoline, safrole and nicotine in OSF fibroblast cultures, respectively. Collagen phagocytosis by fibroblasts exhibited prominent dose-dependent inhibition as the concentration of arecoline, safrole, and nicotine increased. Besides, nicotine had a synergistic effect on arecoline- or safrole-inhibited collagen phagocytosis. CONCLUSIONS: The present study concludes that even in the same person, the collagen phagocytosis by fibroblasts is different between normal and OSF region. The deficiency in collagen phagocytosis by fibroblasts of the lesion might participate in the pathogenesis of OSF. Arecoline, safrole and nicotine, which are released in saliva during BQ chewing plus cigarette smoking, inhibit collagen phagocytosis by fibroblasts in a dose-dependent manner and may induce OSF formation in Taiwan's patients.     

Regulation of interleukin-6 expression by arecoline in human buccal mucosal fibroblasts is related to intracellular glutathione levels

OBJECTIVES: Cytokines play an important role in regulating fibroblast function and is likely to play a key role in regulating the initiation and progression of scarring in any fibrotic disease. Interleukin-6 (IL-6) has been implicated in the development of a variety of fibrotic diseases. The aim of this study was to compare IL-6 expression in fibroblasts cultured from normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce IL-6 expression. METHODS: mRNA level of IL-6 in fibroblasts from OSF was compared with normal buccal mucosa. The effects of arecoline, the major areca nut alkaloid, on IL-6 expression in normal human buccal mucosa fibroblasts (BMFs) were measured in vitro. mRNA was quantified with AlphaImager 2000. To determine whether glutathione (GSH) levels were important in the induction of IL-6 by arecoline, we pretreated cells with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or with buthionine sulfoximine (BSO) to deplete GSH. RESULTS: Fibroblasts derived from OSF exhibited higher IL-6 gene expression than BMF in mRNA levels (P < 0.05). The exposure of quiescent BMF to arecoline resulted in the elevation of IL-6 mRNA expression in a dose-dependent manner (P < 0.05). IL-6 gene regulated by arecoline correlated with intracellular GSH levels in BMF. Arecoline at a concentration of 129 muM induced about 2.7-fold IL-6 mRNA levels over the 6-h incubation period. However, BSO enhanced the IL-6 mRNA levels by 3.9-fold (P < 0.05). In addition, OTZ was found to marginally reduce the arecoline-induced IL-6 expression by about 1.7-fold (P < 0.05). CONCLUSIONS: Taken together, these results suggest that IL-6 expression is significantly upregulated in OSF fibroblasts in areca quid chewers and arecoline may be responsible for the enhanced IL-6 expression. In addition, the regulation of IL-6 expression induced by arecoline is critically dependent on the intracellular GSH concentrations.     

软骨寡聚基质蛋白、趋化因子9和细胞角蛋白19在口腔黏膜下纤维化中的表达

目的 探讨软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)、趋化因子9(C-X-C motif 9,CXCL9)和细胞角蛋白19(keratin 19,KRT19)在口腔黏膜下纤维化(oral submucousfibrosis,OSF)特征性病理变化形成过程中的作用.方法 采用免疫组化技术检测66例OSF和14例正常颊黏膜标本中3种蛋白的表达与定位,同时采用Western blotting和反转录聚合酶链反应(RT-PCR)方法分别在OSF患者和正常颊黏膜组织中检测其蛋白和mRNA的表达.结果 36例(55%)OSF呈现COMP阳性表达,并与嚼槟榔的时间和病理分期有关(P<0.05);CXCL9阳性表达的OSF病例有43例(65%).正常颊黏膜中KRT19均在基底细胞胞质内呈强阳性表达,OSF中只有7例(11%)出现基底细胞KRT19弱阳性表达.Western blotting和RT-PER结果与免疫组化结果一致.结论 COMP、CXCL9和KRT19在OSF发生、发展过程中发挥着重要作用.     

Scanning electron microscopy of different types of oral leukoplakia: Comparison with normal and malignant oral mucosa

The present study analysed surface architecture of normal, premalignant and malignant oral mucosa using scanning electron microscopy to evaluate its role in early diagnosis of potentially malignant oral lesions. The surface ultrastructure of the buccal mucosa in tobacco chewers showed variations from that of non-chewers. Homogenous leukoplakia demonstrated well-defined intercellular junctions and the microrugal surface pattern as seen in normal mucosa. In verrucous leukoplakia, the surface layer consisted of characteristically-shrunken desquamated hyperkeratotic cells. Erosive leukoplakia had a discontinuous superficial layer along with complete loss of intercellular ridges. Speckled leukoplakia also showed marked abnormalities such as thickened irregular protrusions and evidence of a villuslike pattern. These villus-like structures were comparatively prominent in leukoplakia showing dysplasia. Oral carcinoma showed marked altered surface ultrastructure and had a pattern similar to dysplastic lesions. The irregular swollen elongated protrusions with villous-like structures that were observed in carcinoma and dysplastic lesions can, therefore, be considered as surface markers for potentially malignant leukoplakia.     

LncRNA H19与OSF发生及癌变的关系

目的:探讨lncRNA H19在口腔黏膜下纤维性变(OSF)发生及癌变过程中的表达变化及其意义.方法:实时荧光定量PCR技术分别检测12例正常颊黏膜组织、33例OSF颊黏膜组织、31例伴OSF颊癌组织中的LncRNA H19的表达水平.结果:LncRNA H19在颊黏膜组织、OSF颊黏膜组织、伴OSF颊癌组织中的相对表达量分别为1.17±0.37、3.44±1.08、8.88±1.78,组间两两之间比较,P＜0.01.结论:LncRNA H19可能参与OSF的发生及癌变.     

口腔黏膜下纤维化组织中内皮素受体mRNA表达的研究

目的:通过研究内皮素A型受体(endothelin receptor A,ETA)和内皮素B型受体(endothelin receptor B,ETB)在口腔黏膜下纤维化中mRNA的表达,探讨ETA、ETB与口腔黏膜下纤维化(oral submucous fibrosis,OSF)病变发生发展的关系.方法:选取30例OSF患者,其中早、中、晚期组各10例,5例健康志愿者作为对照组,每例研究对象取其口腔颊黏膜,运用逆转录聚合酶链反应(RT-PCR)研究ETA、ETB在OSF中mRNA的表达水平.结果:ETA mRNA和ETB mRNA在OSF各期中的表达均较正常口腔黏膜高(P＜0.05);ETA mRNA在OSF早期的表达明显低于中、晚期(P＜0.05),其中期的表达与晚期相比差异无显著性(P＞0.05);ETB mRNA在OSF各组之间的表达差异无显著性(P＞0.05).结论:咀嚼槟榔等理化因素导致的ETA、ETB在基因转录水平的高表达以及由此所介导的缩血管效应可能是OSF微血管病变发生发展的始动因素.