High prevalence of expression of p53 oncoprotein in oral carcinomas from India associated with betel and tobacco chewing.

A recent study reported a low prevalence of p53 expression (11%) in oral squamous cell carcinomas (SCCs) from South Asia, in contrast to a high prevalence (averaging 52%) in other studies. It was proposed that the different aetiologies for oral SCCs in the South Asia population, i.e. betel and tobacco chewing in combination with smoking and alcohol consumption as compared to smoking and alcohol consumption alone in other populations, may account for the low prevalence of p53 expression. To confirm this hypothesis, we examined p53 expression immunohistochemically in 23 cases of oral SCC from patients in Southern India. Thirteen of the 23 SCCs (56.5%) demonstrated nuclear p53 staining. The expression of p53 was strongly correlated with the number of tobacco-containing quids chewed per day (r = 0.8). These data support the hypothesis that carcinogens derived from tobacco and betel chewing may induce p53 mutations, which in turn are involved in the development of oral cancer.

Response of fibroblast cultures from ataxia-telangiectasia patients to oxidative stress.

The object of this study was to determine whether ataxia-telangiectasia (AT) cells are more sensitive than normal cells to reduced oxygen species generated either during normal cell processes or resulting from metabolism of xenoblotics. To test this hypothesis four AT and four normal fibroblast cultures were exposed to hydrogen peroxide (H2O2) and the induction of micronucleated cells was assayed. AT cultures responded to the H2O2 treatment with a greater increase in micronucleus frequencies than that observed in normal cultures (P less than 0.01). At time course study showed that an elevation in micronucleus frequencies occurred earlier in AT cultures (significant increase by 1.5 h after treatment) than in normal cultures, possibly indicating a G2-phase sensitivity of AT cells to H2O2. The addition of an aqueous extract of areca nut to the cultures, as an example of exogenous stress, induced a greater frequency of micronucleated cells in AT cultures than in the normal cultures. These results suggest that the AT syndrome may serve as a model for investigating the role of reduced oxygen species in cancer.

Variations in the uptake of beta-carotene in the oral mucosa of individuals after 3 days of supplementation.

Variations in the response of individuals to beta-carotene supplementation were studied by measuring the accumulation of beta-carotene in oral mucosa cells. Beta-carotene was administered orally to 178 individuals for 3 consecutive days, exfoliated oral mucosa cells were collected by brushing the entire oral mucosa on the 7th day following supplementation, and the beta-carotene content was measured by HPLC analysis of the pronase-treated cells. The rise in beta-carotene levels in the oral mucosa following supplementation varied considerably. Significant differences in mean beta-carotene levels in the oral mucosa were observed in 4 population groups. After supplementation, beta-carotene levels increased by factors of 10.3 (100 Mile House), 7.8 (Williams Lake), 6.9 (Lytton) and 3.4 (Vancouver), respectively. This difference in mean beta-carotene values is due to there being different proportions of weak and strong responders in the various population groups. Neither peak levels nor increases in beta-carotene levels were correlated with base-line concentrations of beta-carotene in the pre-supplementation samples. A second supplementation was given to 54 individuals several months after the initial supplementation. Of 17 weak responders in the first supplementation study, 10 (58.8%) individuals again showed only a small increase. A time-course study revealed that low responders showed no significant changes in beta-carotene levels over the 21 days following supplementation. Particular attention should be paid to weak responders when results of intervention trials using beta-carotene are interpreted.

Antigenotoxic activity of carotenoids in carcinogen-exposed populations.

Although epidemiological studies suggest the presence of anticarcinogenic agents in the diet, it is difficult to obtain actual proof for the activity of such agents in humans. One approach is to develop and validate potential quantifiable indicators of antigenotoxic/anticarcinogenic agents which can be used in humans belonging to populations at elevated risk for cancer. This paper provides evidence that the exfoliated cell micronucleus test (MEC test) can be used (i) to provide a quantifiable marker for the amount of chromosomal breakage occurring in target tissues of carcinogen-exposed populations; (ii) to indicate the capacity of beta-carotene, alone or in combination with vitamin A, to prevent such damage; and (iii) to predict the response of other biological indicators of cancer risk, such as oral leukoplakias, in individuals receiving oral supplementation with beta-carotene/vitamin A (although the dose and time to response may differ for these endpoints). Future extensions of this approach include establishing the levels of beta-carotene required for antigenotoxic activity in a carcinogen's target tissue by concurrently measuring MEC frequencies and beta-carotene levels in exfoliated cells. In summary, early indications are that the MEC assay is an effective indicator for antigenotoxic agents in carcinogen-exposed individuals and that beta-carotene and vitamin A can suppress such genotoxic activity in at least some populations.

Use of exfoliated cells to study tissue-specific levels of beta-carotene in humans.

This study was designed to explore the feasibility of using exfoliated cells to study beta-carotene incorporation into different epithelial tissues in humans. Exfoliated cells were collected from the oral cavities (by brushing the oral mucosa) and from the urogenital tracts (by centrifuging urine samples) of 36 females and basal levels of beta-carotene (without oral supplementation) were determined. Beta-carotene levels in cells from the two sites differed significantly, although a weak correlation was observed. As a second aspect of the study, 10 of these females were given oral supplementation with beta-carotene (90 mg twice weekly for 4 weeks). Beta-carotene levels increased significantly in both exfoliated urogenital tract (6.8-fold) and oral mucosa (5-fold) cells. However, the supplemented levels remained significantly different for the two types of cells. Beta-carotene levels did not change in individuals receiving a placebo treatment (n = 7). These studies suggest that exfoliated cells collected from different sites may be of value in quantifying tissue levels of beta-carotene during cancer intervention trials.

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.

Use of intermediate endpoints in quantitating the response of precancerous lesions to chemopreventive agents.

A current area of emphasis in cancer research is the determination of whether cancer can be prevented through the use of naturally occurring chemopreventive agents such as beta-carotene. A major area of concern in the design of long-term, large-scale population studies to ascertain the efficacy of such chemopreventive agents lies in the paucity of biological data on the activity of these agents in man. The studies described in this paper were performed to determine whether a series of short-term markers could be used in chemopreventive trials as indicators of the possible success of a chemopreventive regime. Three such markers are described. The first involves the measurement of genotoxic damage in the target tissues of carcinogen-exposed individuals by using the micronucleus test on exfoliated cells. This end point has been successfully used to demonstrate a reduction in carcinogen damage (micronuclei production) in the oral cavity of individuals in population groups at elevated risk for oral cancer (tobacco and betel quid users in the Philippines, snuff users in the Northwest Territories). The second marker involves the determination of DNA adducts in exfoliated cells of carcinogen-exposed individuals by the use of DNA postlabelling procedure. The final marker discussed involves a chemical determination of the levels of a chemopreventive agent in target tissues of individuals receiving a supplement in the diet. In this case, the example described is beta-carotene in exfoliated cells of carcinogen-exposed individuals. These three markers may be combined to determine whether a chemopreventive agent reaches a target tissue, affects DNA adduct formation, and prevents genotoxic damage.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The effect of nass use and smoking on the risk of oral leukoplakia.

The results of a survey of a population with a high incidence of oral cancer are presented in this paper. A remarkably high proportion (41%) of the men surveyed used nass, which is a mixture of tobacco, lime, ash, and cotton oil. The prevalence of oral leukoplakia, lesions thought to be a precursor of oral cancer, was high in persons who used nass (12%) and who smoked cigarettes (15%), and highest among those men who both used nass and smoked (21%). It has been shown that nass use increases the frequency of micronucleated cells in the exfoliated sublingual cells. The clastogenic and genotoxic effect of nass revealed in the study could be attributed to the presence of tobacco-specific N-nitroso compounds. However, the results of chemical analysis, which has shown that nass contains relatively low levels of these compounds, suggest that the relatively strong genotoxic activity of nass could primarily be associated with other chemicals, possibly oxidized phenolics. Despite the uncertainty as to which of the chemicals contained in nass are involved in the etiology of oral cancer and what exactly are the mechanisms of nass-induced carcinogenesis, the results of this study suggest that in populations where nass is widely used, oral leukoplakia and, most probably, oral cancer are conditions that could be prevented by the elimination of nass use and cigarette smoking.

Use of the micronucleus test to monitor the effect of vitamin A, beta-carotene and canthaxanthin on the buccal mucosa of betel nut/tobacco chewers.

The frequency of exfoliated cells with micronuclei in buccal swabs was used to estimate the protective effect of vitamin A, beta-carotene and canthaxanthin (4,4'-diketo-beta-carotene) on the buccal mucosa of betel (areca) nut/tobacco chewers. Micronuclei were scored on exfoliated cells taken by swabbing and stained with the Feulgen reaction and fast green. The betel (areca) nut/tobacco chewers served as their own controls. Prior to the administration of vitamin A and beta-carotene, the examined betel quid chewers had elevated frequencies of micronucleated buccal mucosa cells, averaging 4.03% +/- 1.24 SD (n = 26) and 3.43% +/- 1.22 SD (n = 25), respectively. The frequency of micronucleated buccal mucosa cells in non-chewers and non-smokers was 0.51% (n = 52). Following a 9-week ingestion of vitamin A (150,000 IU/week) and beta-carotene (180 mg/week in 6 capsules), the frequency of micronucleated cells decreased significantly (p less than 0.001) to 1.70% and 1.16%, respectively. No significant shift in the frequencies of micronucleated cells was observed following the intake of canthaxanthin (180 mg/week in 6 capsules) for 9 weeks or that of a placebo. The lack of protective activity of canthaxanthin, which is a good trapper of oxygen singlets but cannot be converted into vitamin A, suggests that vitamin A and beta-carotene exert their inhibitory effect on the formation of micronuclei by a mechanism not involving the scavenging of free radicals. The efficacy of beta-carotene as an inhibitor of micronucleated cell formation, the lack of toxicity, and its availability from a multitude of dietary sources should focus attention on this carotenoid as a promising chemopreventive agent.

Reduction with vitamin A and beta-carotene administration of proportion of micronucleated buccal mucosal cells in Asian betal nut and tobacco chewers.

The frequency of micronuclei in cells scraped from inside the human cheek is a measure of chromosome breakage in earlier cell divisions, and it can be increased tenfold by carcinogenic stimuli. Supplementation for 3 months of the diet of 40 rural Filipino betel chewers with sealed capsules of retinol (100 000 IU/week) and beta-carotene (300 000 IU/week) was associated with a threefold decrease (from 4.2% to 1.4%) in the mean proportion of cells with micronuclei. This proportion decreased in 37 of the 40 supplemented subjects and no large increases were seen in any subjects. In 11 unsupplemented betel chewers in a nearby cluster of houses the mean proportion of micronuclei did not change (4.3% before and 4.8% three months later). This suggests the possibility that in this population an increase in the dietary intake of retinol and/or carotene may reduce the incidence of oral cancer, which is an important neoplasm in many parts of Asia.

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%.

The influence of pH on the convertogenic activity of plant phenolics.

The genotoxicity of plant phenolics, including pyrogallol, gallic acid, resorcinol and catechin, and a water extract and tannin fraction of betel nut (Areca catechu) was examined at pH levels ranging from 5 to 10. Strain D7 of Saccharomyces cerevisiae was used since the cells can withstand a wide range of pH levels without any loss of viability. At alkaline pH ranges, the examined phenolics and betel nut extracts induced mitotic conversion, whereas they lacked this capacity at acid pH levels. This phenomenon may be due to the rapid autoxidation of phenolics under alkaline conditions, which leads to the generation of H2O2 and free radicals. The results indicate that plant phenolics may pose a genotoxic hazard during chewing of lime-containing betel quid and tobacco which causes the salivary pH to rise above 8.

Inhibitory effects of phenolics, teas and saliva on the formation of mutagenic nitrosation products of salted fish.

The objectives of this study were to simulate in vitro some of the conditions that may prevail in man during the ingestion of a meal and to quantitate the inhibitory effect of phenolics and phenolic-containing beverages on the formation of mutagenic nitrosation products. The test system consisted of nitrosating (pH 2, 1 h, 37 degrees C) an aqueous fraction of a salt-preserved Chinese fish (Pak Wik) with or without the inhibitors to be tested and estimating the frequency of his+ revertants per survivor of Salmonella typhimurium (strain TA1535). The phenolics and teas were added to the nitrosation mixture. Catechin, chlorogenic acid, gallic acid, pyrogallol and tannic acid suppressed the formation of mutagenic nitrosation products. The inhibitory efficiency was comparable to that of ascorbic acid. A Japanese, a Chinese and a Ceylonese tea also prevented the formation of mutagenic nitrosated fish products at doses which are usually consumed by man. Moreover, saliva exerted an inhibitory effect. The inhibitory effect was not additive when the phenolics or saliva were added concurrently to the nitrosation mixture. The possibility that phenolics are involved in the apparent chemopreventive effect of fruits and vegetables is discussed.

Inhibition of mutagenicity of a model nitrosation reaction by naturally occurring phenolics, coffee and tea.

Several plant phenolics, one instant coffee, one instant decaffeinated coffee, one roasted coffee, one Japanese tea, one black Indian tea, and one Chinese tea were examined for their inhibitory properties on mutagenicity resulting from the nitrosation of methylurea. Mutagenicity was estimated as the number of his+ revertants per survivor of Salmonella typhimurium TA1535 which was exposed in suspension to the nitrosation mixtures and the modulating agents for 20 min. Tannic acid, gallic acid and chlorogenic acid suppressed the mutagenicity of the model nitrosation system at concentrations similar to or even lower than ascorbic acid. The three tested coffees and three tested teas exerted an inhibitory effect on the mutagenicity of the test system at doses at which they are consumed.

Inhibition of spontaneous mutagenesis in yeast cultures by selenite, selenate and selenide.

Sodium selenite (1--15 mumol/plate) was found to completely suppress spontaneous mutagenesis at 2 independent loci in both wild (YO-300-IC) and mutator (mut 1-1, mut 2-2, mut 3-1, mut 4-1, mut 5-2, mut 6-1, mut 8-1, mut 9-1 and mut 10-1) isogenic strains of Saccharomyces cerevisiae. The 2 loci which where studied were his 1-7, a missense mutation, and lys 1-1, a super-suppressible mutant of the amber variety. The degree of suppression of spontaneous reversion to prototrophy at these 2 loci depended on the concentration of sodium selenite present, the strain of yeast being studied, and the loci being studied. Greater concentrations of sodium selenite (up to 30-fold higher) were required to suppress the frequency of spontaneous reversion at the histidine locus compared to quantities necessary to elicit a similar inhibition of lysine spontaneous reversion rates. The 2 loci also responded differently to the presence of 2 other inorganic selenium derivatives. Spontaneous mutagenesis at the lysine locus for strain YO-800-1C (mu 1-1) was completely inhibited by sodium selenide at 3 mumol/plate with complete suppression of histidine reversion occurring at 30 mumol/plate. Sodium selenate suppressed the spontaneous mutagenesis at the lysine, but not the histidine locus. These results indicate that environmentally added components can have a significant effect on the genetically controlled predisposition of an organism to mutagenesis and suggest the complexity of such interactions.