Changes in DNA copy numbers of bovine papillomavirus type 1 after termination of retinoic acid treatment.

The number of bovine papillomavirus type 1 (BPV) DNA copies [plasmid pdBPV-1 (142-6)] was examined in transformed C127 cells of an RIII mouse during exposure to all-trans-retinoic acid (RA) and after its withdrawal. RA treatment of a transformed cell line reduced the number from approximately 60 copies to an average of less than one copy per cell within 5 weeks. The composition of the RA-treated cell population was heterogeneous with respect to BPV DNA copies: 89.7% of the cells had no detectable copies, 8.6% had one copy, 1.7% had fewer than five copies, and one in 13,000 cells carried more than 10 copies. The low number of BPV DNA copies in the RA-treated cell population did not increase when the cells were subcultured before reaching confluence. RA-treated cell populations that contained less than one BPV DNA copy lost the transformed phenotype. However, a small fraction of cells (1 in 13,000) with greater than or equal to 10 BPV DNA copies retained the capacity to develop into transformed colonies. The relevance of these results to the regression of papillomavirus, DNA-carrying human lesions after exposure to retinoids and the redevelopment of these lesions after withdrawal of retinoids is discussed.

Inhibitory effect of betel nut extracts on endogenous nitrosation in humans.

Extracts of betel nut (Areca catechu) were tested for their capacity to inhibit the endogenous formation of nitrosamines by measurement of the amount of urinary N-nitroso-L-proline (NPRO) following ingestion of sodium nitrate (300 mg) and L-proline (300 mg) by 2 volunteers. A water extract of the dried nuts, an ether extract containing mainly (+)-catechin and (-)-epicatechin, and a caffeine-precipitated n-butyl alcohol extract containing primarily proanthocyanidins (tannins) strongly reduced the endogenous formation of NPRO. An average of 14.7 and 10.9 micrograms NPRO (8 expts per individual) was excreted in the urine of the 2 volunteers over a 24-hour period following the intake of sodium nitrate and L-proline. The water extract and the proanthocyanidin (tannin)-containing extract, both of which contain the dose equivalent of one-quarter of a nut, reduced the excreted NPRO to background levels, which varied from 0.5 to 3.6 micrograms and from 0.6 to 2.1 micrograms (6 expts) in 24-hour urine samples from the 2 volunteers. These results may exemplify the way in which naturally occurring phenolics, which are ingested daily in relatively large quantities, could affect the endogenous formation of carcinogenic nitrosamines.

Inhibitory effect of reducing agents on N-acetoxy- and N-hydroxy-2-acetylaminofluorene-induced mutagenesis.

The effect of cysteine (alpha-amino-beta-mercaptopropionic acid) on the mutagenic activities of the proximate carcinogen, N-hydroxy-2-acetylaminofluorene, and the ultimate carcinogen, N-acetoxy-2-acetylaminofluorene, was examined by estimating the frequency of his+ revertants of Salmonella typhimurium. Nontoxic concentrations of cysteine significantly reduced the formation of revertants when it was applied concurrently with the two carcinogens. Cysteine showed no detectable effect on mutagenesis when added to bacteria before or after exposure to carcinogens. The magnitude of inhibition of mutagenesis depended on the dose of cysteine and the concentration of the carcinogens. Cysteine at equimolar concentrations inhibited to a larger degree the mutagenesis induced by N-hydroxy-2-acetylaminofluorene than it inhibited that elicited by N-acetoxy-2-acetylaminofluorene. The inhibitory action of cysteamine and glutathione was comparable to that of cysteine. The results appear to be consistent with the assumption that cysteine traps electrophiles prior to their action on DNA.

Chromosome-damaging activity of ferritin and its relation to chelation and reduction of iron.

Ferritin from horse spleen was found to cause severe chromosome aberrations in cultured Chinese hamster ovary cells. Ferritin at 15 to 170 microgram/ml was clastogenic and at higher doses was cytotoxic. At comparable concentrations of protein or iron, neither apoferritin nor complexed iron was clastogenic. Sulfhydryl compounds glutathione and cysteine reduced the cytotoxic and clastogenic activities of ferritin. Physiological concentrations of glutathione may normally be sufficient to protect cells from damage. The reducing agent ascorbate had little protective effect. Chelating agents varied in their inhibitory activity: ethylenediaminetetraacetic acid (hexadentate) greater than nitrilotriacetic acid (tetradentate) greater than salicylate (bidentate). 2,2'-Bipyridyl enhance the chromosome-damaging action of ferritin while histidine did not markedly alter the frequencies of aberrations. Catalase and superoxide dismutase showed no inhibitory activity. The mechanism of DNA damage may involve reduction of Fe(III) in the ferritin core to Fe(II), followed by reoxidation of Fe(II) with possible formation of free radicals.

Enhancement of the chromosome-damaging action of ascorbate by transition metals.

Freshly prepared ascorbate inhibited mitosis and induced chromosome aberrations in cultured Chinese hamster ovary cells. Cu(II) and Mn(II) (10(-4) or 10(-5) M) enhanced both actions. Fe(II) and Fe(III) (10(-4) or 10(-5) M) reduced or abolished the mitosis-inhibiting action of ascorbate. At 10(-4) M, Fe(II) and Fe(III) strongly enhanced the chromosome-damaging capacity of ascorbate. Up to 100% of all examined metaphase plates had multiple chromosome exchanges or breaks. Since the cytostatic and clastogenic effect of ascorbate of H2O2 to induce chromosome aberrations was examined. H2O2 and a H2O2: Fe(II) mixture (Fenton reagent) induced chromosome breaks and exchanges but to a lesser degree than did ascorbate: Cu(II), Mn(II), Fe(II), or Fe(III) mixtures. Whether the strong chromosome damaging capacity of ascorbate plus transition metals as seen in the in vitro test system poses a health hazard only properly designed in vivo studies can reveal.

DNA repair synthesis following exposure to chemical mutagens in primary liver, stomach, and intestinal cells isolated from rainbow trout.

DNA repair synthesis was autoradiographically measured in liver, stomach, and intestinal cells isolated from rainbow trout which were exposed in vitro to the chemical mutagens, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline 1-oxide, and aflatoxin B1. The level of repair was greatest in primary hepatocytes which responded to all three mutagens. Only nominal amounts of repair were detected in stomach cells following N-methyl-N'-nitro-N-nitrosoguanidine and 4-nitroquinoline 1-oxide exposures and in intestinal cells following 4-nitroquinoline 1-oxide exposure. In comparison with cultured rainbow trout cells, the quantity of DNA repair found in primary cells is significantly less.

Comparative electrophilicity, mutagenicity, DNA repair induction activity, and carcinogenicity of some N- and O-acyl derivatives of N-hydroxy-2-aminoflourene.

N-Myristoyloxy-N-acetyl-2-aminofluorene, N-acetoxy-N-myristoyl-2-aminofluorene, N-myristoyloxy-N-myristoyl-2-aminofluorene, and N-hydroxy-N-myristoyl-2-aminofluorene each yielded a high incidence of sarcomas in male rats within 5 to 7 months after s.c. injection of 64 micronmoles in divided doses. N-Acetoxy-N-acetyl-2-aminofluorene and N-hydroxy-2-acetylaminofluorene, although potent carcinogens at the s.c. site, were less active than the above derivatives with a myristoyl substituent. N-Sulfonoxy-N-acety--2-aminofluorene (purity larger than or equal to 70%) had little or no carcinogenic activity when administered in large amounts by s.c. injection to rats. The low incidence of tumors could have resulted from N-hydroxy-2-acetylaminofluorene or other decompostion products of the N-sulfonozy derivative. Each of the N-acetoxy and N-myristoyloxy derivatives of N-acetyl-2-aminofluorene and of N-myristoyl-2-aminofluorene showed electrophilic activity toward methionine; N-acetoxy-N-acetyl-2-aminofluorene was the most reactive and N-myristoyloxy-N-myristoyl-2-aminofluorine was the least reactive. Each of these esters also induced unscheduled tritiated thymidine incorportation in nondividing cultured human fibroblasts and thus appeared to induce lesions in DNA that lead to repair synthesis. EACH OF THE N-acetoxy derivatives was highly mutagenic for Salmonella typhimurium strains TA98 and TA1538 without tissue activation; neither N-myristoyloxy derivative was mutagenic under these conditions. While there was a qualitative correspondence between several of the above activities of these 2-aminofluorene derivatives, the quantitative differences and the lack of detectable mutagenicity of the 2N-myristoyloxy derivatives for S. typhimurium indicate the need for multiple short-term tests in the qualitative prediction of potential carcinogenic activity.

Remission of precancerous lesions in the oral cavity of tobacco chewers and maintenance of the protective effect of beta-carotene or vitamin A.

Participants in the intervention trials were fishermen (Kerala, India), who chewed tobacco-containing betel quids daily before and throughout the study period. Frequency of oral leukoplakia, micronuclei in oral mucosal cells, and alterations in nuclear textures were used as endpoints. Administration of vitamin A (60 mg/wk) for 6-mo resulted in complete remission of leukoplakias in 57% and a reduction of micronucleated cells in 96% of tobacco-chewers. beta-carotene (2.2 mmol/wk) induced remission of leukoplakia in 14.8% and reduction of micronucleated cells in 98%. Vitamin A completely suppressed and beta-carotene suppressed by 50% formation of new leukoplakia within the 6-mo trial period. After withdrawal of vitamin A or beta-carotene treatment, oral leukoplakias reappeared, frequency of micronuclei in oral mucosa increased, and nuclear textures reverted to those present before the administration of chemo-preventive agents. The protective effect of the original treatment could be maintained for at least 8 additional months by administration of lower doses of vitamin A or beta-carotene.

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.

Enhancement of bovine papillomavirus-induced cell transformation by tumour promoters.

Cultured C3H/10T1/2 cells transfected with the plasmid pdBPV-1 were used as targets, and the frequency of transformed colonies as the endpoint to test the enhancing capacity of four promoters: 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl-tetradecanoylphorbol-13-acetate (4-O-methyl-TPA), mezerein and phorbol-12-retinoate-13-acetate (PRA). The frequency of the transfected C3H/10T1/2 cells to form transformed colonies was enhanced in the following order: mezerein greater than PRA greater than TPA greater than 4-O-methyl-TPA. The amount of promoters required to promote a tenfold increase in transformed cells was 0.24, 0.81, 30 and 100 ng/ml mezerein, PRA, TPA and 4-O-methyl-TPA, respectively. A significant promoting effect was obtained by a 3.5-day exposure to mezerein regardless of whether it was added at different time intervals after transfection with BPV-DNA. The examined promoters lacked genotoxic activity, as tested on Chinese hamster ovary cells, using chromatid aberrations and exchanges, frequency of macronuclei, unscheduled DNA synthesis (UDS) and inhibition of UDS as endpoints. The usefulness of BPV-1-induced transformation as a bioassay for detecting chemicals with promoting activities is discussed.

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%.

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.

The need for a mammalian test system for mutagens: action of some reducing agents.

Reducing agents and cysteine, cysteamine, glutathione, ascorbic acid and H2O2 with and without the addition of Cu2+ did not increase significantly the frequency of mutations in the Salmonella test at non-toxic concentrations but triggered a marked DNA repair synthesis and induced a relatively high frequency of chromosome aberrations in cultured mammalian cells. Both latter effects were reduced by the addition of catalase to solutions of the reducing agents plus Cu2+. To avoid 'False Negatives' in mutagenicity screening the use of several test subjects including mammalian cells seems to be required.

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.

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.

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.

Tobacco-specific nitrosamines in the saliva of Inuit snuff dippers in the Northwest Territories of Canada.

Levels of tobacco-specific nitrosamines (TSNA), nicotine and cotinine were estimated in the saliva of 20 snuff dippers (Inuit, Northwest Territories, Canada). Levels of N'-nitrosonornicotine (NNN), 4-(methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosoanatabine (NAT) plus N-nitrosoanabiasine (NAB) found in the saliva following a 15-min period of keeping 0.5-1.5 g of moist snuff in the gingival groove are considerable: 115-2610 ppb NNN, 123-4560 ppb NAT + NAB, and up to 201 ppb NNK. The amount of TSNA in the saliva increases with the length of time that the snuff is kept in the mouth. The estimated total amount of 444 micrograms TSNA, the largest part of which will be swallowed, exceeds by far the amounts of nitrosamines ingested through drinking beer (0.34 micrograms/day), eating cured meat products (0.17 micrograms/day), or using cosmetics (0.41 micrograms/day). The relatively high levels of potentially carcinogenic TSNA in the saliva, together with the current popularity of snuff usage by teenagers, is of particular concern.

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.

Mechanism of inhibition of N-methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis by phenolic compounds.

At non-toxic concentrations, 2 naturally occurring phenolic compounds, caffeic acid and chlorogenic acid, suppressed the mutagenic activity of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium strain TA1535. The inhibitory effect was observed only when the phenolic compound and the mutagen were administered concurrently. The interaction between phenolic compounds and MNNG was also studied in a cell-free system using a colorimetric method. The results are consistent with the assumption that phenolic compounds scavenge reactive electrophilic MNNG degradation products, thereby preventing their action on critical cellular targets.

Mutagenic activity of ascorbate in mammalian cell cultures.

Exposure of Chinese hamster ovary (CHO) cells to solutions of ascorbate (2--5 x 10(-4) M) resulted in the induction of somatic mutations at the hypoxanthineguanine phosphoribosyl transferase (HGPRT) locus. Mutant cells were resistant to 6-thioguanine (10 microgram/ml) and sensitive to HAT (hypoxanthine, aminopterin, thymidine) medium. Doses of ascorbate which were mutagenic were also toxic. Addition of catalase to such ascorbate concentrations prevented both mutagenesis and toxicity. This suggests that mutagenic metabolites of ascorbate may involve peroxide radicals.