Elevated frequency of micronucleated cells in the buccal mucosa of individuals at high risk for oral cancer: betel quid chewers

The micronucleus test was applied to buccal mucosa cells of 2 population groups at high risk for oral cancer: Khasis of the northeastern hill region of India, who eat raw betel nuts together with betel leaves and lime, and residents of the state of Orissa (India), who chew betel quids consisting mainly of perfumed tobacco, dried betel nut, betel leaf, lime and several spices. Micronuclei were scored on Feulgen/fast green-stained smear preparations of exfoliated cells obtained by scraping the surface of the buccal mucosa. All 17 raw betel nut eaters and all 20 chewers of betel quids had significantly elevated frequencies of micronucleated mucosa cells over nonchewing controls of comparable ethnic background and dietary habits. The frequencies of micronucleated exfoliated cells were higher at the site within the oral cavity where the quid was kept compared to those at the opposite buccal wall. The micronuclei frequency was lower among individuals chewing a raw betel nut, betel leaf and lime mixture compared to those using tobacco,-betel nut-, lime- and betel leaf-containing quids. Micronuclei frequencies in exfoliated human cells seem to represent a useful 'internal dosimeter' for estimating exposure to genotoxic, and by implication, carcinogenic agents in the tissue from which cancers will develop.     

Piper betel Linn (betel vine), the maligned Southeast Asian medicinal plant possesses cancer preventive effects: time to reconsider the wronged opinion

Since antiquity, Piper betel Linn (betel vine; family Piperaceae) has been an important medicinal agent in the various traditional and folk systems of medicine in Southeast Asia countries. The leaves are the most valued plant part and in the past were routinely used as a chewing agent to prevent halitosis. The leaves are also supposed to harden the gum, conserve the teeth and to prevent indigestion, bronchitis, constipation, congestion, coughs and asthma. Innumerable scientific studies have validated the ethnomedicinal claims. Betel leaves are an integral component of the betel quid that consists of areca nut (Areca catechu Linn.), tobacco (Nicotiana tabacum L) and slaked lime; a highly abused agent with carcinogenic properties. Regular chewing of betel quid is associated mainly with oral cancer and detail studies with individual constituents of the quid have shown that both tobacco and areca nut are carcinogenic, while slaked lime is shown to promote the process of carcinogenesis. However unlike other constituents of the betel quid, the betel leaves devoid carcinogenic effects and on the contrary possesses cancer preventive effects including against the carcinogens present in tobacco. This review for the first time provides information on cancer preventive effects and also addresses the various mechanisms which might be involved.     

Chromosome-damaging activity of saliva of betel nut and tobacco chewers

Saliva of volunteers chewing betel quid, cured betel nut (Areca catechu), betel leaves (Piper betle), a mixture of quid ingredients (dried betel nut flakes, catechu, cardamon, lime, copra and menthol) and Indian tobacco was collected and examined for its genotoxic activity. Chromosome aberrations (chromatid breaks and chromatid exchanges) in Chinese hamster ovary (CHO) cells were used to estimate the genotoxic effect. No detectable levels of clastogenic activity were observed in the saliva of non-chewing individuals. After 5 min of chewing betel quid, betel nut, betel leaves, quid ingredients and Indian tobacco, the saliva samples showed relatively potent clastogenic activities. The addition of transition metals Mn²⁺ and Cu²⁺ to the saliva samples of betel nut and Indian tobacco chewers enhanced their clastogenic activities, whereas Fe³⁺ increased the clastogenicity of the betel nut saliva but decreased the genotoxic effect of the saliva of Indian tobacco chewers. After removal of the betel quid or its components from the mouth, the clastogenic activity disappeared within 5 min. The western-type chewing tobacco did not produce a genotoxic activity in the saliva of chewers. A possible association between the genotoxicity in the saliva of betel quid chewers and the development of oral, pharyngeal and esophageal carcinomas is discussed.     

Promoting activity of betel quid ingredients and their inhibition by retinol

Ingredients of betel quids, which have been linked to the high incidence of precancerous oral lesions and oral cancers, were examined for their promoting activity. Aqueous extracts were tested using the bovine papillomavirus (BPV) DNA transformation assay, which consists of cultured C3H/10T1/2 cells transfected with the plasmid pdPBV-1 as targets, and the frequency of transformed foci as endpoints. Areca nut extracts enhanced the formation of BPV DNA-induced transformed foci approximately tenfold. No promoting activity was detected in two samples of chewing tobacco examined. The addition of retinol to the areca nut extract inhibited its tumour promoting effect in a dose-dependent manner, completely abolishing the promoting activity at a dose of 10(-6) M. The experimental results are compared with epidemiological data on oral cancer incidences among chewers of different areca nut/tobacco mixtures and with the chemopreventive effect of vitamin A administered to betel quid chewers.     

A study of betel quid carcinogenesis. II. Formation of N-nitrosamines during betel quid chewing

In model studies, nitrosation of the major areca alkaloid, arecoline, leads to the formation of N-nitrosoguvacoline, 3-(methylnitrosamino)propionitrile (MNPN), 3-(methylnitrosamino)propionaldehyde and two unknown N-nitrosamines. MNPN is a strong carcinogen in Fischer 344 rats. After subcutaneous injection of 1.1 mmol MNPN in 60 doses, all 15 male and 15 female rats developed tumours within 24 weeks; multiple tumours occurred in 26 of the rats. Eighty-seven percent of the animals had tumours of the oesophagus, 70% had nasal cavity tumours, 37% had tumours of the tongue, 7% tumours of the pharynx and 7% tumors of the forestomach. At the dose used, male and female rats showed no significant difference in tumour incidence or site of tumours. The formation of MNPN during betel quid chewing, although likely, has not yet been proven, while the areca-derived N-nitrosamine, N-nitrosoguvacoline (NG), has been found in the saliva of betel quid chewers at levels of 2.2-348 micrograms/L. N-Nitrosoguvacoline levels were higher in the saliva of chewers who used betel quid together with tobacco. The saliva of these chewers also contained tobacco-specific N-nitrosamines.     

Tobacco (Kretek) Smoking,Betel Quid Chewing and Risk of Oral Cancer in a Selected Jakarta Population

Purpose: This study aimed to determine the association between tobacco consumption (kretek) and betel quidchewing with oral cancer risk. Materials and Methods: A total of 81 cases of oral cancers were matched with162 controls in this hospital-based study. Information on sociodemographic characteristics and details of riskhabits (duration, frequency and type of tobacco consumption and betel quid chewing) were collected. Associationbetween smoking and betel quid chewing with oral cancer were analysed using conditional logistic regression.Results: Slightly more than half of the cases (55.6%) were smokers where 88.9% of them smoked kretek. Afteradjusting for confounders, smokers have two fold increased risk, while the risk for kretek consumers and thosesmoking for more than 10 years was increased to almost three-fold. Prevalence of betel quid chewing among casesand controls was low (7.4% and 1.9% respectively). Chewing of at least one quid per day, and quid combinationof betel leaf, areca nut, lime and tobacco conferred a 5-6 fold increased risk. Conclusions: Smoking is positivelyassociated with oral cancer risk. A similar direct association was also seen among betel quid chewers.     

Role of areca nut consumption in the cause of oral cancers. A cytogenetic assessment.

BACKGROUND. Cytogenetic studies, framed to assess the possible genomic damage caused by areca nut consumption (without tobacco and not as a component of betel quid), were performed among areca nut chewers, which included normal people who chew areca nuts, patients with oral submucous fibrosis, and patients with oral cancer, and healthy nonchewing controls. RESULTS. The analysis showed statistically significant increases in the frequencies of sister chromatid exchanges and chromosome aberrations in peripheral blood lymphocytes and the percentage of micronucleated cells in exfoliated cells of buccal mucosa among all three groups of chewers when compared with those of the controls. CONCLUSIONS. The current data, the first of this type among only areca nut chewers, highlight that this popular masticatory is erroneously considered "safe" and that it increases the genomic damage even when chewed without tobacco. The data also signify that, henceforth, in cytogenetic biomonitoring, areca nut consumption also should be considered as one of the confounding factors.     

Tobacco-specific and betel nut-specific N-nitroso compounds: occurrence in saliva and urine of betel quid chewers and formation in vitro by nitrosation of betel quid

In order to evaluate exposure of betel quid chewers to N-nitrosocompounds, saliva and urine samples were collected from chewersof betel quid with or without tobacco, from tobacco chewers,from cigarette smokers and from people with no such habit, andwere analysed for the presence of N-nitrosamines by gas chromatographycoupled with Thermal Energy Analyzer and alkaloids derived frombetel nut and tobacco by capillary gas chromatography fittedwith nitrogen-phosphorous selective detector. The levels ofthe betel nut-specific nitrosamines, N-nitrosoguvacoline andN-nitrososoguvacine (the latter being detected for the firsttime in saliva), ranged from 0 to 7.1 and 0 to 30.4 ng/ml, respectively.High levels of tobacco-specific nitrosamines were detected inthe saliva of chewers of betel quid with tobacco and in thatof chewers of tobacco, ranging from 1.6 to 59.7 (N'-nitrosonornicotine),1.0 to 51.7 (N'-nitrosoanatabine) and 0 to 2.3 [4-(methyl-nitrosamino)-1-(3-pyridyl)-l-butanone]ng/ml. Urinary concentrations of certain N-nitrosamino acids,including N-nitrosoproline, were determined as a possible indexof exposure to nitroso compounds and their precursors in thestudy groups: no clear difference was observed. The betel nut-specificalkaloid, arecoline, was present at high levels in the salivaof betel quid chewers with or without tobacco. Nicotine andcotinine were also detected in saliva and urine of chewers oftobacco and of betel quid with tobacco. In order to assess whetherN-nitroso compounds are formed in vivo in the oral cavity duringchewing or in the stomach after swallowing the quids, the levelsof N-nitroso compounds in betel quid extracts were determinedbefore and after nitrosation at pH 7.4 and 2.1. The resultsindicate that N-nitroso compounds could easily be formed invivo. The possible role of N-nitroso compounds in the causationof cancer of the upper alimentary tract in betel quid chewersis discussed.     

Ortho- and meta-tyrosine formation from phenylalanine in human saliva as a marker of hydroxyl radical generation during betel quid chewing

The habit of betel quid chewing, common in South-East Asia andthe South Pacific islands, is causally associated with an increasedrisk of oral cancer. Reactive oxygen species formed from polyphenolicbetel quid ingredients and lime at alkaline pH have been implicatedas the agents responsible for DNA and tissue damage. To determinewhether hydroxyl radical (HO) is generated in the human oralcavity during chewing of betel quid, the formation of o- andm-tyrosine from L-phenylalanine was measured, Both o- and m-tyrosinewere formed in vitro in the presence of extracts of areca nutand/or catechu, transition metal ions such as Cu²⁺ and Fe²⁺and lime or sodium carbonate (alkaline pH). Omission of anyof these ingredients from the reaction mixture significantlyreduced the yield of tyrosines. Hydroxyl radical scavengerssuch as ethanol, D-mannitol and dimethylsulfoxide inhibitedthe phenylalanine oxidation in a dose-dependent fashion. Fivevolunteers chewed betel quid consisting of betel leaf, arecanut, catechu and slaked lime (without tobacco). Their saliva,collected after chewing betel quid, contained high concentrationsof p-tyrosine, but no appreciable amounts of o- or m-tyrosine.Saliva samples from the same subjects after chewing betel quidto which 20 mg phenylalanine had been added contained o- andm-tyrosine at concentrations ranging from 1010 to 3000 nM andfrom 1110 to 3140 nM respectively. These levels were significantlyhigher (P< 0.005) than those of subjects who kept phenylalaninein the oral cavity without betel quid, which ranged from 14to 70 nM for o-tyrosine and from 10 to 35 nM for m-tyrosine.These studies clearly demonstrate that the HO radical is formedin the human oral cavity during betel quid chewing and is probablyimplicated in the genetic damage that has been observed in oralepithelial cells of chewers.     

Effect of lime composition on the formation of reactive oxygen species from areca nut extract in vitro

Lime, representative of that used by betel quid chewers, was collected in a region of Papua New Guinea where the incidence of oral cancer is high. The free calcium hydroxide content and pH of 25 lime samples were highly correlated with the generation of reactive oxygen species from areca nut extract in vitro, and DNA damage in vitro, measured as 8-hydroxy-2'-deoxyguanosine. Fe2+ and Mg2+ levels in the lime samples were too low to modify formation of reactive oxygen species, but hydrogen peroxide formation was almost entirely inhibited by addition of Mg2+ to the reaction mixture. These results suggest that the calcium hydroxide content of lime in the presence of areca nut is primarily responsible for the formation of reactive oxygen species which might cause oxidative damage in the DNA of buccal mucosa cells of betel quid chewers.     

A review of betel quid chewing, oral cancer and precancer in Mainland China

On the Chinese mainland, betel quid (BQ) chewing is common in the Hunan and Hainan provinces. The BQ chewing habit in Hunan consists of dried husks and betel nuts, which are sold as industrially packaged, areca nut-based products. In Hainan, the fresh nut is chewed. Tobacco is not added. Reported prevalence of BQ chewing in Hunan province is high (64.5-82.7%). Oral diseases associated with BQ chewing are oral submucous fibrosis (OSF), oral leukoplakia (OL) and oral cancer. Reported prevalence of OSF among BQ chewers ranges from 0.9% to 4.7%. People most commonly affected are between the ages of 30 and 39 years, and 40 and 49 years. The reported prevalence of OL in Hainan ranges from 2.1% to 2.5%. In BQ chewers who also smoke, the reported prevalence is 20.3%. The prevalence of OL in Hunan province ranges from 0.1% to 0.5%. The prevalence of oral cancer among BQ chewers is low, ranging from 0.02% to 0.05%. In cases of OSF, reported prevalence is 2.6% and 1.2%. Presently, data on prevalence of BQ chewing in southern provinces of Mainland China is limited. BQ chewing habits, however, seem to differ between geographic areas. Future case-control studies are necessary to evaluate the risk for oral cancer and other associated oral mucosal diseases resulting from variations in BQ chewing habits.     

Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells

Because betel quid chewing has been linked to the development of oral cancer, pathobiological effects of an aqueous areca nut extract, four areca nut alkaloids (arecoline, guvacoline, guvacine, and arecaidine), and four nitrosated derivatives [N-nitrosoguvacoline, N-nitrosoguvacine, 3-(N-nitrosomethylamino)propionaldehyde and 3-(N-nitrosomethylamino)propionitrile] have been investigated using cultured human buccal epithelial cells. Areca nut extract in a dose-dependent manner decreases cell survival, vital dye accumulation, and membrane integrity, and it causes formation of both DNA single strand breaks and DNA protein cross-links. Depletion of cellular free low-molecular-weight thiols also occurs, albeit at quite toxic concentrations. Comparisons of the areca nut-related N-nitroso compounds and their precursor alkaloids, at concentrations up to 5 mM, indicate that 3-(N-nitrosomethylamino)propionaldehyde is the most potent on a molar basis to decrease both survival and thiol content and to cause significant formation of DNA single strand breaks. Arecoline, guvacoline, or N-nitrosoguvacoline decreases survival and cellular thiols, whereas arecaidine, guvacine, N-nitrosoguvacine, and 3-(N-nitrosomethylamino)propionitrile have only minor effects on these variables. Taken together, the present studies indicate that aqueous extract and, in particular, one N-nitroso compound related to areca nut, i.e., 3-(N-nitrosomethylamino)propionaldehyde, are highly cytotoxic and genotoxic to cultured human buccal epithelial cells, of potential importance in the induction of tumors in betel quid chewers.     

A study of betel quid carcinogenesis. 1. On the in vitro N-nitrosation of arecoline

Betel quid chewing is strongly associated with cancer of theoral cavity. Extracts of betel quid are tumorigenic in the experimentalanimal, but thus far, not a single carcinogen has been detectedin the tobacco free quid. This study is based on the hypothesisthat during chewing, arecoline, the major alkaloid of the betelnut, gives rise to carcinogenic N-nitros-amines. In vitro experimentsreported here have shown that N-nitrosation of arecoline leadsto N-nitrosoguvacoline (NG), 3-(methylnitrosamino)propionitrile(MNPN) and 3-(methyI-nitrosoamino)propionaldehyde. Although,according to an earlier study, NG is most likely not carcinogenic,MNPN is suspected to be a relatively strong animal carcinogenbased on bioassays with its lower homologue. The conditionsprevailing in the oral cavity of betel quid chewers are likelyto favor the formation of these three nitrosamines.     

Betel quid not containing tobacco and oral cancer: a report on a case-control study in Papua New Guinea and a meta-analysis of current evidence

Smoking and betel quid chewing are associated with increased risk of oral cancer but few studies have reported on associations in populations where betel quid does not contain tobacco. We conducted a case-control study in Papua New Guinea and a systematic review. Our case-control study recruited 143 cases with oral cancer and 477 controls. We collected information on smoking and betel quid chewing. Current smoking was associated with an increased risk of oral cancer with an adjusted odds ratio (OR) for daily smokers of 2.63 (95% confidence intervals (95% CI) 1.32, 5.22) and amongst heaviest smokers of 4.63 (95% CI 2.07, 10.36) compared to never-smokers. Betel chewing was associated with increased risk of oral cancer with an adjusted OR for current chewers of 2.03 (95% CI 1.01, 4.09) and in the heaviest chewers of 2.47 (95% CI 1.13, 5.40) compared to nonchewers. The OR in those who both smoked tobacco and chewed betel quid was 4.85 (95% 1.10, 22.25), relative to those who neither smoked nor chewed. The systematic review identified 10 previous studies that examined risk of oral cancer associated with betel quid chewing that controlled for smoking in populations where betel quid did not contain tobacco. In studies that reported results for non-smokers the combined OR was 2.14 (95% CI 1.06, 4.32) in betel quid chewers and in studies that adjusted for smoking the combined OR was 3.50 (95% CI 2.16, 5.65) in betel quid chewers. Preventive efforts should discourage betel quid chewing as well as smoking.     

Cytotoxic Effects of Betel Quid and Areca Nut Aqueous Extracts on Mouse Fibroblast,Human Mouth-Ordinary-Epithelium 1 and Human Oral Squamous Cell Carcinoma Cell Lines

Background: Betel quid chewing is more common among the older generation in rural areas of Malaysia. Oral cancer in Asia has been associated with the habit of chewing betel quid and areca nut. Objective:  This study aims to investigate the cytotoxic effects of betel quid and areca nut extracts on the fibroblast (L929), mouth-ordinary-epithelium 1 (MOE1) and oral squamous cell carcinoma (HSC-2) cell lines. Methods: L929, MOE1 and HSC-2 cells were treated with 0.1, 0.2 and 0.4 g/ml of betel quid and areca nut extracts for 24, 48 and 72 h. MTT assay was performed to assess the cell viability. Results: Both extracts, regardless of concentration, significantly reduced the cell viability of L929 compared with the control (P<0.05). Cell viability of MOE1 was significantly enhanced by all betel quid concentrations compared with the control (P<0.05). By contrast, 0.4 g/ml of areca nut extract significantly reduced the cell viability of MOE1 at 48 and 72 h of incubation. Cell viability of HSC-2 was significantly lowered by all areca nut extracts, but 0.4 g/ml of betel quid significantly increased the cell viability of HSC-2 (P<0.05). Conclusion: Areca nut extract is cytotoxic to L929 and HSC-2, whereas the lower concentrations of areca nut extract significantly increased the cell viability of MOE1 compared to the higher concentration and control group. Although betel quid extract is cytotoxic to L929, the same effect is not observed in MOE1 and HSC-2 cell lines. Further investigations are needed to clarify the mechanism of action.     

The precancer risk of betel quid chewing,tobacco use and alcohol consumption in oral leukoplakia and oral submucous fibrosis in southern Taiwan

In areas where the practise of betel quid chewing is widespread and the chewers also often smoke and drink alcohol, the relation between oral precancerous lesion and condition to the three habits is probably complex. To explore such association and their attributable effect on oral leukoplakia (OL) and oral submucous fibrosis (OSF), a gender-age-matched case-control study was conducted at Kaohsiung, southern Taiwan. This study included 219 patients with newly diagnosed and histologically confirmed OL or OSF, and 876 randomly selected community controls. All information was collected by a structured questionnaire through in-person interviews. A preponderance of younger patients had OSF, while a predominance of older patients had OL. Betel quid chewing was strongly associated with both these oral diseases, the attributable fraction of OL being 73.2% and of OSF 85.4%. While the heterogeneity in risk for areca nut chewing across the two diseases was not apparent, betel quid chewing patients with OSF experienced a higher risk at each exposure level of chewing duration, quantity and cumulative measure than those who had OL. Alcohol intake did not appear to be a risk factor. However, cigarette smoking had a significant contribution to the risk of OL, and modified the effect of chewing based on an additive interaction model. For the two oral premalignant diseases combined, 86.5% was attributable to chewing and smoking. Our results suggested that, although betel quid chewing was a major cause for both OL and OSF, its effect might be difference between the two diseases. Cigarette smoking has a modifying effect in the development of oral leukoplakia.     

Betel quid and oral cancer: prospects for prevention.

Betel-quid chewing is an ancient and socially accepted practice. The introduction of tobacco reinforced this practice, and now almost all habitual chewers of betel quids include tobacco. It is well established that chewing of betel quid with tobacco causes oral cancer and is largely responsible for the high incidence of oral cancer in several South Asian countries. The feasibility of primary prevention of oral cancer was studied in a population-based prospective intervention study. A cohort of 12,212 betel-quid chewers and smokers was exposed to a programme of health education for stopping chewing and smoking and subjected to annual examinations for detection of oral precancerous lesions. Evaluations after one, five and eight years showed that primary prevention of oral cancer is feasible and practicable. Early detection of oral cancer is an important control measure. In a secondary prevention study, 53 basic health workers were trained in the detection and referral of lesions suspected of being oral cancer. Over one year, they examined more than 39,000 high-risk individuals, resulting in the detection of 20 cases of oral cancer. The sensitivity and specificity of their diagnoses was assessed through a re-examination of a 5% sample: we concluded that it was possible to incorporate a secondary prevention programme into the existing health care system.     

Endogenous nitrosation in the oral cavity of chewers while chewing betel quid with or without tobacco

In order to evaluate endogenous nitrosation in the oral cavity of chewers of betel quid with tobacco (BQT) or without tobacco (BQ), saliva samples were collected from healthy male volunteers after chewing sequentially (i) unmodified BQT or BQ, (ii) BQT or BQ to which proline has been added, and (iii) BQT or BQ to which proline and ascorbic acid had been added. Samples were collected over 20 min and analysed for N-nitrosoproline (NPRO), tobacco-specific nitrosamines (TSNA) and areca nut-specific nitrosamines using gas chromatography-thermal energy analysis, arecoline and nicotine using gas chromatography-nitrogen phosphorus-specific detector, and for nitrite and thiocyanate. When results were expressed as a ratio of NPRO (ng/ml) to nicotine (micrograms/ml), all BQT chewers had increased NPRO contents after chewing BQT with proline. For BQ chewers, when the results were expressed as a ratio of NPRO (ng/ml) to arecoline (micrograms/ml), a similar increase in NPRO content was observed. However, the presence of ascorbic acid inhibited the increased nitrosation in only four out of ten BQT chewers and in five out of ten BQ chewers; in the rest of the samples, its presence enhanced the levels of NPRO. N'-Nitrosoanatabine (NAT) and N-nitrosoguvacoline (NGCO) levels decreased significantly in saliva of chewers of BQT in the presence of ascorbic acid, suggesting inhibition of their formation. In-vitro nitrosation of BQT/BQ with proline and proline plus ascorbic acid showed a similar pattern of nitrosation at salivary pH. The study confirmed previous results that certain nitrosamines are formed during the chewing of BQT/BQ.     

Prevention of Betel Quid Chewers’ Oral Cancer in the Asian-Pacific Area

“Betel quid chewers’ oral cancer” is one of the most common malignancies in South and SoutheastAsian countries.Oral premalignancies are also very common in betel quid chewers and about 10% of these undergo malignanttransformation. Although education for cessation of the betel quid chewing habit is important, there are few adequatestrategies and policies for primary prevention, health promotion and education related to oral cancer control, especiallyin rural areas. In addition to oral health education, it is also crucial to establish a data-management system as wellas monitoring and evaluation systems for oral cancer prevention.     

Angiotensin-converting enzyme gene insertion/deletion polymorphism is associated with risk of oral precancerous lesion in betel quid chewers

To investigate whether angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism is related to the risk of oral precancerous lesions (OPL) in Taiwanese subjects who chew betel quid, a total of 61 betel quid chewers having OPL were compared with 61 asymptomatic betel quid chewers matched for betel quid chewing duration and dosage. The frequency of homozygote for ACE D variant is significantly higher in the case subjects than that of the controls (44.3 vs 24.6%; P = 0.0108). The adjusted odds ratio of the D homozygous for the risk of OPL is 8.10 (95% confidence interval (CI) = 2.04-32.19, P = 0.003). In the allelic base analysis, the D allele is also significantly associated with higher risk of OPL. When grouping the study subjects by smoking status, the association between ACE I/D polymorphism and risk of OPL was only observed in nonsmokers. Our results support the theory that genetic factors may contribute to the susceptibility of OPL and suggest that smoking and genetic factors may be differently involved in the development of OPL.