Association between lysyl oxidase polymorphisms and oral submucous fibrosis in older male areca chewers

Objective:  Areca use is the major cause for oral squamous cell carcinoma and oral submucous fibrosis (OSF) in South Asians. Lysyl oxidase (LOX) is a copper-activated enzyme critical for collagen cross-linking and organization of extracellular matrix. The presence of a G to A polymorphism at nucleotide 473 caused a non-conservative Arg158Gln change in the LOX amino acid sequence. OSF is a precancerous lesions characterized by the accumulation of collagen in oral submocousa. The aim of this study was to investigate the relationship between LOX Arg158Gln polymorphism and the risk of OSF.
Method:  PCR-restriction fragment length polymorphisms and direct sequencing was utilized to compare LOX polymorphic allelotype in male areca-chewing controls ( n  = 216) and OSF ( n  = 83) patients.
Results:  There was a borderline of statistically significant difference in Arg158Gln genotype lying between control and OSF patients. However, the G/A+A/A of LOX Arg158Gln in OSF patients older than 50 year was statistically significantly higher than controls older than 50 year (odd's ratio: 4.48; 95% CI = 1.58–12.67).
Conclusion:  The elder OSF patients were increased in LOX Arg158Gln. Our findings may suggest a potential application in risk population selection using LOX polymorphism for preventive intervention of OSF genesis in a subset of areca chewers.     

Etiology of oral submucous fibrosis with special reference to the role of areca nut chewing

Oral submucous fibrosis (OSF) is a high risk precancerous condition, predominantly affecting Indians. Consumption of chilli was hypothesized as an etiologic factor on the basis of ecological observations and a solitary animal experimental study. Subsequent epidemiologic studies that included case-series reports, large cross-sectional surveys, case-control studies, cohort and intervention studies have identified areca nut as the major etiologic agent. Tissue-culture studies involving human fibroblasts, areca nut extracts and areca nut alkaloids supported this etiologic hypothesis by showing fibroblastic proliferation and increased collagen formation. Currently, the role of genetic susceptibility and that of autoimmunity are receiving attention. The influence of nutritional factors, if any, remains unclear.     

The fibroblast population in oral submucous fibrosis

The purpose of the investigation was to compare the morphology of fibroblasts cultured from healthy oral mucosa and mucosa of patients with oral submucous fibrosis (OSF) and to collate the occurrence of cell types of similar morphology. Cells cultured from biopsy specimens from the buccal mucosa of six subjects who did not chew the areca nut and six patients with OSF who chewed areca nut were grown according to standard techniques. Ninety cells per cell line were recorded daily for 8 days, classified into types F1, F2 and F3 according to their morphology, and the results statistically analyzed. We found that there was a relative increase of F3 cells in relation to Fl cells in OSF resulting in the ratio of F3 to F1 cells being significantly larger in OSF than the ratio in the controls. As it has been reported that F3 cells m rat connective tissues produce significantly more collagen types I and III than F1 cells, we concluded that a change of fibroblast population has occurred in OSF and that this relative increase of F3 cells in humans, which could be committed to the production of large quantities of collagen, can be an explanation for the excessive collagen formation in OSF.     

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口腔黏膜下纤维性变(oral submucous fibrosis,OSF)是一种慢性、隐匿性且具有癌变倾向的疾病,主要发生于印度、巴基斯坦等东南亚国家以及我国湖南、台湾两省。OSF病因不明,咀嚼槟榔是其最主要的致病因素,且咀嚼槟榔的频率越高、年限越长,越易患OSF。所有OSF患者都有咀嚼槟榔史,咀嚼槟榔还与口腔白斑、口腔癌发病高度相关。此外,OSF的发生还与遗传因素、免疫反应、胶原相关性基因,营养缺乏等有关。本文就OSF致病因素做一简单介绍。     

Growth of oral and skin fibroblasts from patients with oral submucous fibrosis

The purpose of the investigation was to evaluate and compare the proliferation (growth) of mouth fibroblasts and skin fibroblasts from patients with oral submucous fibrosis (OSF). Material comprised fibroblasts from fibrous bands situated in the buccal mucosa and from the inner aspect of the forearm of 8 patients with classic features of OSF as well as fibroblasts from 6 buccal mucosa and 8 skin biopsy specimens from healthy non-areca nut chewing individuals. Cells were cultured for 8 days according to standard techniques. Their growth was monitored daily, under optimal conditions as well as exposure to concentrations of arecoline. The data were analyzed using regression analysis, analysis of variance and the Kruskal-Wallis test. We found no statistically significant differences between the proliferation patterns of oral and skin fibroblasts from patients or between those from patients and controls. The reaction of the cells exposed to concentrations of arecoline was similar; at low concentrations (0.1–10 μg/ml) normal growth was maintained, while 100 μg/ml inhibited growth. It is concluded that fibroblasts from mouths affected by OSF have proliferation patterns which fall within normal parameters, that the excessive collagen formation in established OSF is not due to increased fibroblast proliferation and that arecoline does not stimulate fibroblast proliferation.     

HLA-antigens in oral submucous fibrosis

HLA–typing was carried out on 122 areca nut chewers who attended hospitals for complaints unrelated to the habit. The subjects were South Africans of Indian extraction. The study did not include haplotypes. Palpable fibrous bands in the mouth indicated oral submucous fibrosis. The subjects were divided into 4 groups based on specific oral symptoms and signs. Groups A and B were without fibrous bands. Group A (47 subjects) included those with one or no symptoms while group B (28 subjects) suffered from 2 to 7 oral symptoms. Group C (17 subjects) had oral symptoms and represented early or mild oral submucous fibrosis and exhibited at least one discrete palpable fibrous hand. Group D (30 subjects) were classic oral submucous fibrosis cases with multiple bands. The high occurrence of oral submucous fibrosis in this study group (39%) is similar to the occurrence in comparable age groups reported earlier–n South Africa and is conceivably due to the higher age range of the subjects and their relatively long exposure to the area nut. We were unable to demonstrate a specific pattern of HLA–antigen frequencies in chewers with or without the disease. Furthermore, there were no differences between the study population and the controls. It is concluded that there is not necessarily a HLA–associated susceptibility in oral submucous fibrosis.     

Predictors of the severity of oral submucous fibrosis among gutka consumers: a regression analysis

Oral submucous fibrosis (OSMF) is an insidious chronic disease of the oral mucosa that is characterised by severely limited mouth opening, blanching of the oral mucosa, and a burning sensation in the oral cavity. Consumption of betel nut and/or gutka are the known risk factors. We undertook this study to correlate the frequency and duration of gutka intake with the severity of OSMF and to determine the predictors of severe OSMF (mouth opening <20mm). A cross sectional study was conducted on 300 participants (who were known gutka chewers) selected at the Baqai Dental College and Fatima Hospital, Karachi, Pakistan. Participants’ medical and dental histories were recorded. Informed consent was obtained, and clinical oral examination was done. Information regarding the extent of mouth opening, chewing habits, frequency and duration of gutka intake, the site of placing gutka, duration of chewing, and whether they swallowed or spat out the gutka were collected. A standardised questionnaire was used to document the findings. Binary logistic regression was applied using the severity of OSMF (mouth opening <20mm) as an outcome variable. Out of 300 participants, 172 (57.3%) were males; mean (SD) age of the sample was 38.2 (12.3) years. A total of 156 (52%) participants had the habit of chewing gutka, of which 213 (71%) had clinical stage I OSMF, 75 (25%) had stage II, and 12 (4%) had stage III. As per functional staging, 18 (6%) subjects had mouth opening <20mm. Nearly 144 (48%) participants were consuming other forms of tobacco in addition to the gutka. Patients with palpable bands = 232 (77.3%), ulcerative lesions = 212 (70.7%), altered taste sensation = 210 (70%) and altered hunger = 252 (85.7%) were common findings. The duration of gutka intake was found to be positively correlated with the severity of OSMF; however; its frequency was not. Among intraoral findings, the presence of red and white lesions inside the mouth was the most significant predictor of the severity of OSMF.     

槟榔提取物刺激口腔黏膜角朊细胞培养上清对成纤维细胞增殖活性的影响

目的：探讨槟榔提取物刺激口腔黏膜角朊细胞在黏膜下纤维性变发病中的作用。方法：采用不同浓度槟榔提取液刺激体外培养的角朊细胞,取细胞培养上清,MTT法观察细胞培养上清对成纤维细胞增殖的影响。结果：一定浓度槟榔提取液刺激的角朊细胞培养上清能促进成纤维细胞增殖;细胞培养上清对成纤维细胞增殖的促进作用存在个体差异。结论：槟榔成分可能通过改变口腔黏膜角朊细胞的活性而导致口腔黏膜下纤维性变的发生。     

Aloe vera in treatment of oral submucous fibrosis: A systematic review and meta‐analysis

The management of oral submucous fibrosis (OSF) is quite challenging, and as yet, no effective therapy is available for its management. The present systematic review and meta‐analysis sought to assess the effectiveness of aloe vera in alleviating pain and clinical signs of OSF. A comprehensive search in PubMed/Medline, Scopus, and Web of Science databases was conducted to identify the relevant randomized clinical trials. RevMan 5.3 software was used for data analysis. Six randomized controlled trials fulfilled the inclusion criteria. The results of meta‐analysis showed statistically significant differences between aloe vera and control groups in alleviating pain/burning sensation at the end of the first and second month, in favor of aloe vera, but no significant differences were found at the end of the third month. With regard to objective clinical outcomes, no statistically significant differences were found between the groups. Aloe vera has a promising effect in reducing pain/burning sensation and clinical improvement in patients with OSF. However, owing to the marked heterogeneity of the included studies, conducting well‐designed trials with long follow‐up periods, standardized outcome measures and adequate sample sizes are warranted.     

Prevalence and characteristics of areca nut chewers among junior high school students in Changhua county, Taiwan

Abstract – Some studies indicate that betel quid and its ingredients chewing can produce cell mutagenicity and tumorigenicity. In Taiwan studies, betel quid chewing is the main cause of submucous fibrosis and oral cancer. Understanding the distribution and characteristics of the areca nut chewing population is one of the first steps in the effort to prevent these oral diseases. A stratified cluster random sample of 2442 junior high school students in Changhua county, Taiwan, were surveyed for the habit of areca nut chewing. Significantly more male students chewed areca nut than female students (9.2% vs 0.9%). The proportion of students who were chewing areca nuts increased with increasing (seventh to ninth) grades. Areca nut was used by junior high school students at a higher rate in village (rural) areas as compared to town (semi-urban) and city (urban) areas (6.4%, 3.7% and 3.0%, respectively). More students in the ordinary achievement classes were chewing areca nuts than those in the high achievement classes (8.4% vs 1.6%). Areca nut chewing students tended to have users in their families. Cigarette smoking and alcohol drinking were positively associated with areca nut chewing. More than half (53.6%) of the areca nut chewing students first experimented with this habit with a family member, most often the father or grandfather.     

Role of areca nut in the causation of oral submucous fibrosis: a case-control study in Pakistan

A case-control study on chewing and smoking habits and oral submucous fibrosis (OSF) was undertaken in Karachi in 1989/90. Cases were patients detected with fibrous bands and leathery mucosa and hospital-based controls were matched by age and sex. Information on habits was collected by personal interview of 157 cases and 157 controls. Despite overall female preponderance, a substantial number of young men were enlisted. The male/female risks were found to be similar. Immigrants from India to Pakistan (Mohajir) had a similar risk status to local Punjabis. No differences between risks were found when comparing the three age categories. 21.21-40, 41-60 yr. Among the cases, an increased risk was observed for areca nut chewing. This habit when practised alone appeared to have the highest risk (RR 154), followed by pan with or without tobacco (RR 64. 32 respectively). Logistic regression and discriminant analysis showed that daily consumption rates appeared to be more important with respect to risk than lifetime duration of habit. Tobacco habits were more prevalent amongst those 15 cases who presented with concurrent carcinoma and OSF: We conclude that areca nut chewing has a causal relationship with OSF: additional tobacco insult may be necessary for subsequent carcinoma development.     

Development of an in vivo mouse model to study oral submucous fibrosis

BACKGROUND: Epidemiological data have shown an association of areca nut chewing with oral submucous fibrosis (OSF). Experimental evidence to confirm this has been limited. Fibrosis-promoting activity of areca nut was tested in an animal model. METHOD: Buccal mucosa of a group of 20 female BALB/c strain mice, 10-12 weeks of age, was treated twice daily 6 days per week with topical application of aqueous areca nut extracts for 300-600 days. A control group (n = 20) was treated with 50 mM NaCl. The influence of areca nut on the oral epithelium and connective tissue was recorded semiquantitatively by light microscopy. RESULTS: The areca nut-treated oral epithelium showed progressive changes in epithelial thickness leading to atrophy, increased cellularity of fibroblasts, fibrosis of connective tissue, focal infiltration of inflammatory cells and muscle atrophy. On killing after 600 days of treatment, the scores on cellularity, inflammation and muscle atrophy were significantly different to the control group (P = 0.03). CONCLUSION: The study provides further evidence that areca nut contributes to the development of OSF in treated animals. The model has the potential to test synergism of areca nut with other carcinogens and any therapeutic interventions.     

槟榔与细胞内活性氧及自噬的关系

槟榔作为一级致癌物,其与口腔癌的关系受到广泛关注。活性氧（ROS）及自噬水平与肿瘤的发生发展密切相关,研究发现槟榔可诱发细胞内ROS及自噬水平变化。本文结合相关研究进展,对槟榔、细胞内ROS以及自噬之间的关系进行综述。     

Pilot survey of oral mucosa in areca (betel) nut chewers on Hainan Island of the People''s Republic of China

100 people on Hainan Island were studied for their smoking and chewing habits and the condition of their oral mucosa. Ninety-five percent of the study population chewed areca (betel) nut. In men this habit was supplemented with either smoking cigarettes or water pipe. In two men a small commissural leukoplakia was found. In three women clinical and histologic changes pointed towards oral submucous fibrosis.     

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.     

Psychiatric morbidity among patients with oral sub‐mucous fibrosis: a preliminary study

J Oral Pathol Med (2010) 39 : 761–764 Background: Oral submucous fibrosis (OSF) is a chronic oral mucosal condition with pre‐malignant potential. The association between psychiatric morbidity and OSF has been sparsely studied. Methods: This cross‐sectional study included 33 consecutive subjects clinically diagnosed with OSF who attended an academic tertiary care dental centre. General Health Questionnaire‐28 was administered to assess the psychological morbidity. Dependence to areca products was assessed using the ICD‐10 criteria. Patient group was compared with an age and gender matched control group (n = 33) from the same hospital that comprised patients who had dental diseases other than OSF and had no areca/tobacco habit. Results: Total mean scores (SD) of GHQ‐28 for study group and control group respectively were 48.2 (18.1) vs. 24.2 (6.5) (P < 0.001). Among the patient group, the mean total GHQ scores were significantly higher in those with functional stage C (those with worst functional staging) of OSF 63.4 (15.2) when compared with those with stage B 54 (14.2) and stage A 38.7 (17) (d.f. = 2; P = 0.005). 22 (66.7%) of patients met ICD‐10 criteria for dependence to areca products. Conclusions: OSF may be associated with considerable psychological morbidity. This morbidity may be significantly more in patients with worst functional staging. It is difficult to determine whether this is the cause or effect of the disease. Further systematic studies are urgently needed to address this issue.     

Effect on the incidence of oral submucous fibrosis of intervention in the areca nut chewing habit

Incidence of oral submucous fibrosis was calculated from a 10-yr prospective intervention study of 12,212 individuals with a strong component of health education on tobacco and area nut chewing. Based on 11 new cases among 6341 chewers, the annual incidence was 8.0 per 100,000 among men and 29.0 for women. An earlier 10-yr follow-up study, with no intervention component, served as control. Based on 11 new cases among 3,809 chewers, the annual incidence was 21.3 per 100,000 for men and 45.7 for women controls. Although the decrease in the incidence in the intervention cohort was not statistically significant due to small number of cases, the results underscored the causal role of areca nut chewing and indicated the potential for primary prevention of oral submucous fibrosis.