Micronuclei in exfoliated human cells as a tool for studies in cancer risk and cancer intervention

The use of the micronucleus test on exfoliated cells as an approach to identify genotoxic damage in human tissues which are targets for organ-specific carcinogens and from which carcinomas will develop, is described. Chromosomal damage by carcinogens to dividing basal cells of the epithelium results in the production of micronuclei in the daughter cells which migrate up through the epithelium and are exfoliated. Exfoliated cells can be readily obtained from several tissues, including the oral buccal mucosa (scrapings of oral cells), bronchi (sputum), urinary bladder and ureter (centrifugation of urine), cervix (smears) and esophagus (imprints from biopsies). The micronucleus test on exfoliated cells has been successfully used to: (1) recognize population groups at an elevated risk for cancer of the oral cavity or urinary bladder; (2) estimate synergistic or additive effects of carcinogen exposure (cigarette smokers plus drinkers of alcoholic beverages); (3) pinpoint the site within an organ from which most carcinomas will develop (oral cancers among 'inverted' smokers in the Philippines). The possibility that this assay may also serve as a rapid monitor for chemopreventive agents is suggested by a preliminary trial on the effect of vitamin A/beta--carotene dietary supplementation among 33 betel quid chewers in the Philippines. These individuals received sealed capsules of retinol (100,000 IU/week) and beta-carotene (300,000 IU/week) for a 3-month period. At the end of this time, the frequencies of micronucleated buccal mucosa cells were reduced from an average of 4.2% to 1.4%. No changes were observed in micronucleus frequencies among 11 betel quid chewers not receiving vitamin pills. Non- chewers of betel quid in this population had a micronucleus frequency of 0.5%.     

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.     

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.     

Relationship between cellular levels of beta-carotene and sensitivity to genotoxic agents

The usefulness of an in vitro test system to predict the inhibitory effect of beta-carotene on the genotoxic activity of carcinogens/mutagens was explored. To facilitate the comparison of data obtained from cultured cells (CHO) and from exfoliated human cells, endpoints were used which can be quantitated in both cell systems: the frequency of micronuclei for estimating the effect of genotoxic agents, and cellular levels of beta-carotene as a protective agent. In CHO cells, beta-carotene inhibited the clastogenic and micronucleus-forming effect of methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4NQO), but had no protective action against gallic acid, tannic acid, and aqueous extract of areca nut or H2O2. The extent of inhibition depended on the ratio of beta-carotene to MMS. Doses of beta-carotene which exerted a protective effect in vitro ranged from approximately 2 to 5 ng per 10(6) CHO cells. Comparable levels of beta-carotene were previously found to reduce the frequency of micronucleated exfoliated cells from the buccal mucosa of tobacco and areca-nut chewers (Stich et al., 1984b).     

Oral lesions, genotoxicity and nitrosamines in betel quid chewers with no obvious increase in oral cancer risk

A link between the generation of areca nut-related N-nitrosamines in the saliva, the induction of genotoxic damage in the oral mucosa, as judged by an increase in micronucleated exfoliated cells (MEC), and a low incidence of oral cancer was studied in 2 population groups characterized by their habit of chewing quids without tobacco: Guamanians, who chew areca nuts (Areca catechu) with or without the addition of betel leaf (Piper betle); Taiwanese, who use areca nut, betel leaf or inference and slaked lime. The levels of N-nitrosoguvacoline (NG) in the saliva of chewers of fresh green areca nuts were very high (70.8 ng/ml) as compared to those reported for individuals using the more complex Indian betel quids (0.91 ng/ml or 5.6 ng/ml). None of the other areca nut-related nitrosamines (N-nitrosoguvacine (NGC), 3-(methylnitrosamino)propionitrile (MNPN) and 3-(methylnitrosamino)propionaldehyde (MNPA)) were detected in the saliva of Taiwanese betel quid chewers. The addition of slaked lime to the areca nut enhances the formation of NG during a chewing session. The frequency of MEC did not increase in the oral mucosa of areca nut chewers who do not use slaked lime, but showed a small but significant elevation in individuals using lime-containing quids. The elevation of MEC in Taiwanese, who are at low risk for oral cancer, is relatively small as compared to that found in chewers of Indian betel quids (pan), who show a highly elevated oral cancer risk. The results seem to suggest that NG may play only a minor role, if any, in the etiology of oral cancer among betel quid chewers.     

Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with beta-carotene and with beta-carotene plus vitamin A

Fishermen from Kerala (India) who chewed tobacco-containing betel quids daily (17.2 +/- 9.6 quids per day) and had well-developed oral leukoplakias with elevated frequencies of micronucleated cells participated in a short-term intervention trial. Beta-carotene (180 mg/week) (Group I), beta-carotene (180 mg/week) plus vitamin A (100,000 IU/week) (Group II), and placebo (Group III) capsules were given twice weekly for 6 months under strict supervision. The remission of oral leukoplakias, the inhibition of new leukoplakias, and the reduction of micronucleated oral mucosal cells were recorded at the 3rd and 6th months of the trial period. After 3 months, the frequency of micronucleated cells was significantly reduced in Group I (from 4.09% to 1.1% in areas of leukoplakia, and from 4.1% to 1.0% in the normal mucosa). At this time, remission of oral leukoplakias did not differ significantly from that observed in the placebo group. After 6 months of treatment, remission of leukoplakias in Group I (14.8%) and Group II (27.5%) differed significantly from that seen in Group III (3.0%). The development of new leukoplakias during the 6-month period was strongly inhibited in Group II (7.8%), and to a lesser degree in Group I (14.8%), as compared to Group III (21.2%). During the trial period, all participants continued to chew tobacco-containing betel quids in their accustomed manner. Thus, remission and inhibition of new oral leukoplakias and reduction of micronucleated mucosal cells occurred in the groups receiving beta-carotene and beta-carotene plus vitamin A during the continuous presence of carcinogens derived from tobacco and areca nut.     

Remission of oral precancerous lesions of tobacco/areca nut chewers following administration of beta-carotene or vitamin A, and maintenance of the protective effect

Designs of intervention trials are based on results from a multitude of disciplines. In this study, a comparative analysis of various chewing mixtures used by groups from different geographical locations (Guam, Peru, Taiwan, the Philippines, and India) and their link to oral cancer incidences was used to trace the ingredients responsible for oral carcinogenesis among chewers. The usefulness of applying intermediate endpoints in intervention trials was examined by comparing the response of micronucleated mucosal cells and oral leukoplakia of chewers of tobacco-containing betel quids to the twice weekly administration of beta-carotene (180 mg/week), vitamin A (100,000 IU/week or 200,000 IU/week), and beta-carotene (180 mg/week) plus vitamin A (100,000 IU/week). A reduced frequency of micronucleated mucosal cells and remission of leukoplakias resulted following a 3- to 6-month treatment. The development of new leukoplakias was also inhibited. The various endpoints differed in degree and time course to the administration of beta-carotene and vitamin A. Following termination of the beta-carotene or vitamin A administration, micronucleated cells and leukoplakia recurred in the oral cavity of chewers who continued this habit throughout the trial period. Attempts were made to maintain the protective effect achieved by the treatment with relatively high doses of the chemopreventive agents. Vitamin A given at a level of 50,000 IU/week was able to keep the frequency of micronucleated mucosal cells at low levels for at least a 12-month post-treatment period, whereas beta-carotene administered at 60 mg/week was less effective in maintaining the protective effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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 pilot beta-carotene intervention trial with Inuits using smokeless tobacco

The frequency of exfoliated cells with micronuclei (MNC) was used to estimate the genotoxic effect of smokeless tobacco (snuff) on the oral mucosa and to follow the response to the administration of beta-carotene (180 mg/week, given twice weekly in 6 capsules of 30 mg each). The pilot trial was carried out with Inuits in Gjoa Haven, Northwest Territories, Canada. Their traditional diet, which is rich in caribou and seal meat and liver but low in vegetables and fruits, leads to "normal" serum levels of retinol (447 ng/ml in non-users of tobacco and 463 ng/ml in tobacco users) but low levels of beta-carotene (57 ng/ml for non-users of tobacco and 47 ng/ml for users). Prior to the twice-weekly administration of beta-carotene, the frequency of MNC was 1.87% +/- 0.92 (n = 23) in the mucosa of the lower gingival groove where the tobacco was usually kept. It decreased significantly (P less than 0.001) to 0.74% +/- 0.42 following the 10-week oral administration of beta-carotene capsules. The frequency of MNC did not change significantly in the group receiving a placebo and in snuff users who received no treatment over the 10-week trial period. The size and morphological appearance of the typical snuff-related, whitish, wrinkled patches of the mucosa where the tobacco was kept was not affected by the 10-week treatment with beta-carotene. Similarly, no reduction was observed in the frequency of anucleated, exfoliated mucosa cells. Beta-carotene appears to be an efficient inhibitor of MNC in the oral mucosa of snuff users who do not suffer from any vitamin A deficiency and who have "normal" levels of retinol.     

Response of oral leukoplakias to the administration of vitamin A

Tobacco/betel nut chewers (Kerala, India) with well-developed oral leukoplakias were chosen for a short-term intervention trial of vitamin A therapy. Participants were randomly distributed into two groups, one receiving 200,000 IU vitamin A per week (0.14 mg/kg body wt/per day) for 6 months, and the other receiving placebo capsules. Their cancer-causing habit, which can be quantitated (an average of 13.1 betel quids/day, 26.1 min/quid), did not change during the trial period. The 6-month oral administration of vitamin A caused complete remission in 57.1% of participants, and a total suppression of the development of new leukoplakias in all chewers receiving vitamin A (n = 21), as compared to 3% and 21%, respectively, in the placebo group (n = 33). The results were substantiated by examining the histological and cytological changes on small biopsies which were taken at the onset and at the completion of the trial period. Over the 6-month period of vitamin A administration, the number of layers of spinous cells decreased in 85% of the participants, the loss of polarity of basal cells was reduced from 72.2% to 22.2% of chewers, subepidermal lymphocytic infiltration was greatly diminished from 66.7% to 5.5% of chewers, and nuclei with condensed chromatin disappeared from the epidermal layer (72.2% before to 0% at the end of the trial).     

Quantitating the synergistic effect of smoking and alcohol consumption with the micronucleus test on human buccal mucosa cells

The micronucleus test was applied to exfoliated cells of the buccal mucosa of four population groups: (A) non-smokers and non-drinkers of alcoholic beverages, (B) non-smokers but alcohol drinkers, (C) smokers but non-drinkers, and (D) smokers and drinkers. An elevated frequency of micronucleated buccal mucosa cells was observed only in group D (smokers and alcohol drinkers). When group D was subdivided according to the number of cigarettes smoked, the frequency of micronucleated buccal cells and the average number of micronuclei per cell appeared to depend upon cigarette consumption. An approximately eight-fold increase of micronucleated mucosa cells was seen among alcohol drinkers who smoked three or more packs of cigarettes per day, and an approximately 4.2-fold elevation was observed when one to two packs were consumed. Neither smoking alone of up to and over 60 cigarettes per day nor ethanol drinking alone of up to 1.21 per day led to a detectable elevation of micronucleated buccal mucosa cells. Whether the strong synergistic effect between smoking and alcohol consumption, as seen by the frequency of micronucleated buccal mucosa cells, is related to their synergistic effect in the induction of oral cancers is an intriguing but open question.     

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water- based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well- established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.      

Khat (Catha edulis) consumption causes genotoxic effects in humans

We used the micronucleus (MN) test to determine the genetic damage caused by khat, a widely consumed psychostimulant plant, in exfoliated cells of volunteers who chewed the drug on a regular basis. In the first study in which we compared the frequency of MN in buccal and bladder mucosa cells in 20 khat consumers (10-160 g/day) and 10 controls, a pronounced (8-fold) increase in micronucleated buccal mucosa cells was seen among khat consumers; khat consumption did not lead to a detectable elevation of micronucleated bladder mucosa cells. Among heavy khat chewers, 81% of the MN had a centromere signal indicating that khat is aneuploidogenic. To investigate the effect of simultaneous consumption of tobacco and alcoholic beverages, we compared the MN frequency in buccal cells of 25 khat consumers (20-85 g/day) who smoked cigarettes (15-60/day) and drank alcoholic beverages (15-80 g of pure ethanol/day) with a control group (control group I) of 25 individuals matched for age, body weight, tobacco and alcohol consumption and with another control group of 25 individuals (control group II) not consuming any of the drugs. The frequency of buccal mucosa cells with MN was higher in control group I than in group II and the effect of khat, tobacco and alcohol was found to be additive. A time-kinetics study on khat-induced MN showed that the highest frequency of MN was observed during the fourth week after consumption. In light of the large body of evidence on the close association between genetic damage and cancer, these results suggest that khat consumption, especially when accompanied by alcohol and tobacco consumption, might be a potential cause of oral malignancy.     

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.     

Monitoring of sister chromatid exchange and micronuclei as biological endpoints

Cytologically visible damage in human chromosomes can be detected as structural chromosomal aberrations, numerical changes in genome, sister chromatid exchanges (SCE) or as micronucleated cells. The importance of in-vivo cytogenetic damage that is induced in human cells is that it indicates that similar alterations may have occurred in other tissues, either in somatic or in germinal cells. SCEs represent symmetrical exchanges between sister chromatids; generally, they do not result in alteration of the chromosome morphology or the genetic information. Although the detection method is highly sensitive as an in-vitro screening test, in monitoring studies, it seems to be restricted to cases where the exposing agents are strong alkylating compounds (e.g., ethylene oxide, cytostatic drugs) or to some multi-exposure conditions (e.g., cigarette smoking, laboratory work, rubber industries). Micronuclei arise from acentric chromosome fragments or lagging whole chromosomes. They have been detected in a variety of dividing human cells, including exfoliated epithelial cells, bone-marrow cells and lymphocytes. Positive responses of induction of cells with micronuclei have been obtained in studies with cells of the buccal mucosa (chewers of betel or tobacco) or cultured lymphocytes of some groups occupationally exposed to agents like styrene or ethylene oxide.     

Beta-carotene levels in exfoliated human mucosa cells following its oral administration

Beta-carotene levels of exfoliated oral mucosa cells can be increased severalfold by the oral administration of this provitamin. Beta-carotene was estimated by HPLC analysis in pronase-treated exfoliated cells obtained by brushing the entire oral mucosa with a toothbrush. A small percentage of individuals did not respond with an increase of beta-carotene in their mucosa cell in spite of a relatively large intake of the provitamin (360 mg in 4 days, or 2880 mg in 16 weeks, respectively). Levels of beta-carotene in the mucosa cells are affected by the concurrent administration of vitamin A: 0.27 ng beta-carotene per 10(6) cells in the placebo group, 1.79 ng following the intake of beta-carotene (180 mg/week for 16 weeks), and 4.29 ng after beta-carotene (180 mg/week for 16 weeks) plus vitamin A (100,000 IU/week for 16 weeks) consumption. The considerable variations in tissue levels of beta-carotene following its oral administration must be taken into account when cancer intervention trials using this agent are designed and evaluated.     

Effect of riboflavin, retinol, and zinc on micronuclei of buccal mucosa and of esophagus: a randomized double-blind intervention study in China

A randomized double-blind intervention trial was done in Huixian, People's Republic of China, a population with a high incidence of esophageal cancer. The aim of the trial was to determine whether a once-a-week treatment with retinol (15 mg or 50,000 IU), riboflavin (200 mg), and zinc (50 mg) could result, after 1 year, in a lower prevalence of precancerous lesions of the esophagus in the group receiving the active treatment as compared with the prevalence in the group receiving a placebo. The results of the trial, published elsewhere, indicated that the treatment had no effect on the prevalence of precancerous lesions of the esophagus. In determining whether an effect could be detected when earlier end points are used, the prevalence of micronuclei was evaluated in exfoliated cells from the esophagus and from the buccal mucosa in the present study. In a subsample of 200 out of the original 610 study subjects, smears were taken from the buccal mucosa before and after treatment, and in 170 subjects esophageal smears were obtained during endoscopy only after treatment. The smears were fixed and kept at room temperature over 1 year before being evaluated for the presence of micronuclei by means of 4'-6-diamidino-2-phenylindole fluorescent staining. Smears from approximately half of the subjects were considered suitable for evaluation. No statistically significant difference in the prevalence of micronuclei in the buccal mucosa cells was observed before and after treatment (the mean percentage of micronucleated cells in the vitamin group upon first examination, before treatment started, 0.35%; 1 year after treatment, 0.31%) or between the treatment and the placebo group at the final examination. (The mean percentage of micronucleated cells in the vitamin-treated group was 0.31 and 0.39% in the placebo group.) However, a statistically significant reduction (P = .04) was observed in the prevalence of micronuclei in esophageal cells in the treatment group as compared to the placebo. (The mean percentage of micronucleated cells in the vitamin-treated group was 0.19%; it was 0.31% in the placebo group.