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.

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.

Mutagenicity of fecal extracts from carnivorous and herbivorous animals.

Extracts of the feces of 3 carnivorous animals (dog, river otter and sea gull) and 5 herbivorous animals (cow, horse, sheep, chicken and goose) induced chromosome aberrations (breaks and exchanges) in cultured CHO cells. The addition of CuII (10(-4)M) enhanced the clastogenic effect of fecal extracts of the examined animals with the exceptiion of 1 dog and 3 cow samples. Catalase reduced the chromosome-breaking and mitosis-inhibiting capacities of fecal extracts. These results indicate the presence of hydrogen peroxide-forming compounds. The possibility must be considered that animal and human excreta may be a major source of mutagens entering man's environment.

Potentiation of genotoxicity by concurrent application of compounds found in betel quid: arecoline, eugenol, quercetin, chlorogenic acid and Mn2+.

5 components of the betel quid were examined for their clastogenic activities individually and in various combinations. They included the alkaloid, arecoline, from the betel nut (Areca catechu L.), eugenol, from the betel vine (Piper belle L.), chlorogenic acid, from tobacco leaves (Nicotiana tabacum), quercetin, from fennel seeds (Foeniculus vulgare Mill.) and the ubiquitous transition metal Mn2+. The clastogenic effects of the concurrent applications of arecoline plus eugenol, arecoline plus quercetin and arecoline plus chlorogenic acid were greater than the sum of the action of each individual component. Similarly, the combinations of arecoline, chlorogenic acid and Mn2+ induced frequencies of chromosome aberrations which exceeded the sum of the clastogenic activities of individually applied compounds or the sum of the clastogenic activities of 2 jointly applied compounds (arecoline plus Mn2+, or chlorogenic acid plus Mn2+). The clastogenic activity was estimated as the frequency of metaphase plates with at least 1 chromatid break or chromatid exchange, or the average number of chromatid breaks and exchanges per Chinese hamster ovary (CHO) cell. A potentiating (enhancing) action was also evident when 2 clastogens were used at doses which would not lead to a detectable increase in the frequency of chromosome aberrations when applied individually. It may be useful to distinguish between a "genotoxic range", which would be applicable to individually assayed compounds, and a "cogenotoxic range", which may include concentrations at which a chemical exerts a potentiating effect when combined with other genotoxic or non-genotoxic compounds.

Clastogenic activity of caramel and caramelized sugars.

Cultured Chinese hamster ovary (CHO) cells were exposed for 3 h to caramelized solutions of the sugars sucrose, glucose, mannose, arabinose, maltose and fructose. Each of these caramelized sugars induced a relatively high frequency of chromosome breaks and exchanges in the treated cells. The non-caramelized sugars did not increase the frequency of chromosome aberrations. A potent clastogenic effect was also observed when a commercially used caramel powder was assayed. Up to 54% of all examined metaphase plates of the treated CHO cells had at least one chromosome break or exchange. This chromosome-damaging action of commercial caramel powder was reduced in the presence of liver microsomal (S9) preparation or FeII and FeIII. The transition metals CuII and MnII neither enhanced nor reduced the clastogenic activity of the caramel powder.

Differential sensitivity of Xeroderma pigmentosum cells of different repair capacities towards the chromosome breaking action of carcinogens and mutagens.

The sensitivity of cultured fibroblasts obtained from four unrelated Xeroderma pigmentosum patients (XP-K, XP-C, XP-E and XP-H), which showed different DNA repair levels, was examined. The frequency of metaphase plates with chromosome aberrations and the frequency of breaks and exchanges per chromosome complement were estimated following exposure to the carcinogens 4-nitroquinoline-1-oxide (4NQO),N-acetoxy-2-acetyl-aminofluorene (N-acetoxy-2-AAF), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and to the mutagen daunomycin. The frequency of chromosome aberrations (breaks and exchanges) increased in the order (XP-K less than XP-C less than XP-E less than XP-H) with decreasing DNA repair capacity of the XP cells examined (XP-K greater than XP-C greater than XP-E greater than XP-H) following 4NQO and N-acetoxy-2-AAF. MNNG induced DNA repair synthesis and chromosome aberrations in the four XP cell types at levels comparable to those in fibroblasts of non-afflicted persons. Daunomycin triggered no DNA repair synthesis but induced similar frequencies of chromosome aberrations in the XP cells and controls. Heterozygous XP cells from parents of XP-K, XP-E and XP-C responded as control cells towards the three carcinogens and the mutagen used. Xeroderma pigmentosum can be considered to be an "induced" chromosome instability syndrome, in contrast to Bloom's syndrome or Fanconi's anaemia, which are "spontaneous" chromosome breakage syndromes according to German's definition.

[New method of thrombocyte counting. Technic of electronic counting of low thrombocyte concentrations in peripheral blood]. / Neue Methode zur Thrombozyten-Zählung. Methode zur elektronischen Zählung niedriger Thrombozyten-Konzentrationen des peripheren Blutes

Starting from recent studies of Balleisen (1972) and Schmidt et al. (1973) about automated counting of platelets an automated method has been developed for accurate platelet counting at very low platelet concentration of peripheral blood. The method can be summarized as follows: 0,1 ml anticoagulated venous blood (Na-EDTA or polystyrol tubes containing EDTA powder) or capillary blood from finger tips was diluted with 20 ml of a mixture of Isoton-Periston-(Bayer-)Na2-EDTA (22:2:1 vol.) in polystyrol tubes. After 5 min centrifugation at 85 g, using an angle head and decantation into a polystyrol tube, second centrifugation. Decantation in a Coulter glass and counting at aperture 8, attenuation 0.500 and theshold 6 respectively 70 using at 70 mikron capillary. Counts at threshold 70 (erythrocyte counts) were subtracted from counts at threshold 6 (platelets and erythrocytes); to calculate platelet counts/mm3 multiplication by factor 2 at undiluted venous or capillary blood. The mean variation coefficient of our method being 4,1%, counting of platelets is possible with the accuracy of other automated methods at normal platelet concentration. At very low platelet counts our method seems to be the most accurate laboratory method for platelet counting.

Adhesive-fixed partial dentures in anterior and posterior areas. Results of an on-going prospective study begun in 1985.

Within the framework of an on-going prospective clinical study begun in 1985, 120 adhesive-fixed partial dentures (AFPD) continued to be examined. The manufacture and the fitting of the AFPDs were carried out following a standard procedure. The preparation technique and the metal framework conditioning (silica-coating, sand-blasting and electrochemical etching) has varied throughout the duration of the study. Using Kaplan-Meier analysis, the survival rate was determined and an analysis of risk with regard to location factors (anterior, posterior; maxilla, mandible), conditioning and preparation techniques (retentive/non-retentive) was determined using the Cox regression model. The location of the AFPD had no influence on the survival rate. The survival time was determined mainly by the preparation technique. Strict preparation of seating grooves and pin holes made a 95% survival rate possible after 10 years (Kaplan-Meier estimation). Without retention, the risk of failure increased by a factor of 3.7.

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.