Induction of chromosome instability and stomach cancer by altering the expression pattern of mitotic checkpoint genes in mice exposed to areca-nut.

BACKGROUND: There are strong indications for a causal association between areca-nut consumption and cancers. In Meghalaya, India, the variety of areca-nut is used as raw and unprocessed form whose chemical composition and pharmacological actions have been reported. Yet we know little on the initial pathway involved in areca-nut associated carcinogenesis since it is difficult to assess its effects on genetic alterations without interference of other compounding factors. Therefore, present study was undertaken in mice to verify the ability of raw areca-nut (RAN) to induce cancer and to monitor the expression of certain genes involved in carcinogenesis. This study was not intended to isolate any active ingredients from the RAN and to look its action. METHODS: Three groups of mice (n = 25 in each) were taken and used at different time-points for different experimental analysis. The other three groups of mice (n = 15 in each) were considered for tumor induction studies. In each set, two groups were administered RAN-extract ad libitum in drinking water with or without lime. The expression of certain genes was assessed by conventional RT-PCR and immunoblotting. The mice were given the whole RAN-extract with and without lime in order to mimic the human consumption style of RAN. RESULTS: Histological preparation of stomach tissue revealed that RAN induced stomach cancer. A gradual increase in the frequency of precocious anaphase and aneuploid cells was observed in the bone marrow cells with a greater increment following RAN + lime administeration. Levels of p53, Bax, Securin and p65 in esophageal and stomach cells were elevated during early days of RAN exposure while those of different mitotic checkpoint proteins were downregulated. Apoptotic cell death was detected in non-cancerous stomach cells but not in tumor cells which showed overexpression of Bax and absence of PARP. CONCLUSION: Present study suggested (a) RAN induces stomach cancer, however, presence of lime promoted higher cell transformation and thereby developed cancer earlier, (b) perturbations in components of the chromosome segregation machinery could be involved in the initial process of carcinogenicity and (c) the importance of precocious anaphase as a screening marker for identification of mitotic checkpoint defects during early days.

Proteomics-based identification of plasma biomarkers in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is an aggressive cancer and its occurrence is closely related to betel nut chewing in Taiwan. However, there are few prognostic and diagnostic biomarkers for this disease especially for its association with betel nut chewing. Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of OSCC. In present study, plasma samples from OSCC patients with at least 5-year history of betel nut chewing and healthy donors were analyzed by fluorescence 2D-DIGE-based proteomic analysis. Totally, 38 proteins have been firmly identified representing 13 unique gene products. These proteins mainly function in inflammatory responses (such as fibrinogen gamma chain) and transport (Apolipoprotein A-I). Additionally, the current quantitative proteomic approach has identified numerous OSCC biomarkers including fibrinogen (alpha/beta/gamma) chain, haptoglobin, leucine-rich alpha-2-glycoprotein and ribosomal protein S6 kinase alpha-3 (RSK2) which have not been reported and may be associated with the progression and development of the disease. In summary, this study reports a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers in OSCC. The potential of utilizing these markers for screening and treating OSCC warrants further investigations.

Severity of periodontal disease in individuals chewing betel quid with and without tobacco.

BACKGROUND: The deleterious effects of chewing betel quid (BQ) with or without tobacco on periodontal health are poorly addressed. The aim of this study was to investigate the severity and extent of periodontal disease among individuals chewing BQ with and without tobacco. METHODS: One hundred twenty individuals (70 BQ chewers: 35 with tobacco and 35 without tobacco) and 50 control individuals (non-chewers) were included in this study. Sociodemographic data and information regarding BQ chewing habit were collected using a questionnaire. Plaque index, bleeding on probing and probing pocket depth were measured. Numbers of missing teeth were recorded and marginal bone loss was measured on panoramic radiographs. Statistical analyses were performed using 1-way analysis of variance and Bonferroni post hoc test. RESULTS: The socioeconomic status of subjects in the control group was significantly higher as compared with those chewing BQ either with or without tobacco. Plaque index, bleeding on probing and probing pocket depth were greater in subjects chewing BQ with tobacco than in those chewing BQ without tobacco and the controls. Subjects chewing BQ with tobacco had fewer teeth than those chewing BQ without tobacco and the controls. Marginal bone loss was higher in subjects chewing BQ with tobacco than in those chewing BQ without tobacco and the controls. CONCLUSIONS: The severity of periodontal disease is enhanced in subjects chewing BQ with tobacco as compared with those chewing BQ without tobacco. Subjects with a low socioeconomic status and poor education are significantly more likely than others to develop periodontal disease.

Toxicological evaluation of Tetracarpidium conophorum nut oil-based diet in rats.

The effects of Tetracarpidium conophorum nut oil-based diet on the growth performance and some biochemical constituents of rat tissues was investigated following a feeding period of 6 weeks. The results revealed that the volume of water taken, the amount of feed consumed and the weight gained by the animals maintained on the nut oil-based diet were not significantly (P>0.05) different from those fed on soybean oil-based diet. The reduction in the activities of ALP, GOT and GPT in the liver and heart of animals fed on the nut oil-based diet was accompanied by increase in the serum enzymes. The nut oil-based diet significantly reduced (P<0.05) serum concentrations of total cholesterol and HDL-C whereas triglycerides and atherogenic index increased. The serum LDL-C level of the nut oil-based diet fed animals compared well with those of soybean oil-based diet. These alterations suggested that adverse effects have occurred, possibly by altered membrane permeability of the hepatocytes and cardiac cells. Similar alterations in the serum lipids of animals maintained on nut oil-based diet also portends cardiovascular risk. Although, T. conophorum nut oil did not adversely affect growth performance and the feeding appetite of the animals, it is not completely 'safe' for consumption.

Involvement of the mitochondrion-dependent pathway and oxidative stress in the apoptosis of murine splenocytes induced by areca nut extract.

Areca quid chewing is a major risk factor for oral submucous fibrosis and oral cancer. Clinical evidence suggests that the pathophysiology of the oral diseases is closely associated with immune deterioration. The objective of the present studies was to investigate the pro-apoptotic effect of areca nut extract (ANE) in lymphocytes. Exposure of naïve splenic lymphocytes to ANE significantly enhanced apoptosis in a time- and concentration-dependent manner. Results from Hoechst staining confirmed the morphological features characteristic of apoptosis in ANE-treated cells. ANE treatment induced the depolarization of mitochondrial membrane potential (Deltapsi(m)), which preceded the occurrence of apoptosis. In parallel with the disruption of Deltapsi(m), ANE induced the release of cytochrome c, and the activation of caspase-9, indicating the activation of the mitochondrion-dependent pathway. Moreover, an increased level in the intracellular reactive oxygen species was detected in ANE-treated lymphocytes undergoing apoptosis. ANE-mediated apoptosis, caspase-9 activation and ROS production, but not Deltapsi(m) depolarization, were partially but significantly attenuated in the presence of the antioxidant N-acetyl-L-cysteine (NAC). Collectively, these results demonstrated the pro-apoptotic effect of ANE in primary lymphocytes, which was mediated, at least in part, by the activation of the mitochondrion-dependent pathway and oxidative stress.

A review on grain and nut deterioration and design of the dryers for safe storage with special reference to Turkish hazelnuts.

Turkey produces about 80% of the total hazelnut crop of the world. About 75% of the production are exported. In Turkey hazelnuts are traditionally sun dried, and may be subject to mold growth and subsequent mycotoxin formation due to prolonged drying time under humid and rainy weather conditions. Drying hazelnuts in a reasonable time after harvest is necessary for mycotoxin-free, high-quality products. In general, nuts and cereals contaminated by the toxins pose a potential hazard not only to the people of the producer countries, but also to people of the importing countries, if they should be regarded as safe by inefficient sampling plans, therefore preventing toxin formation actually benefits very large populations. Deterioration and health hazards associated with toxin contaminated hazelnuts and other nuts and cereals have similar causes and consequences; therefore, deterioration of the nuts and cereals in storage has been reviewed by considering as many grains and nuts as possible, then special reference was made to hazelnuts. Proper preharvest practices followed by proper drying and safe storage reduces the hazards associated with contamination by the toxins. This article reviews the pre- and post-harvest practices, and the grain- and nut-drying systems required for toxin-free products. Because drying is the major unit operation involving this process, the drying systems and the mathematical models required for their design is also discussed.

[A trial of the preparation Cheblin-SK-1 in models of nematodiases]. / Ispytanie preparata Cheblin-SK-1 na modeliakh nematodozov.

A kerosene milky-stage walnut (Juglans spp.) extract, a folk medication, has come into wide use in the past 30 years. The drug CK-I was prepared on a scientific basis. Its acute toxicity and toxicological profile were studied on albino mice and rats, chickens, chicken embryos, piglets. The maximum non-lethal dose of CK-I was 19 g/kg for albino mice and 21 g/kg for albino rats. The drug can be classified as i.v. hazard class. The anthelmintic effects of CK-I were examined in mice with cyphaciasis and in chickens with ascariasis and heterakiasis. In murine cyphaciasis, CK-I given in a dose of 75 mg/kg to albino mice provided 100% efficiency. Its doses of 800 and 1000 mg/kg were required to achieve this effect in chick ascariasis and heterakiasis, respectively.

Aflatoxin variability in pistachios.

Pistachio fruit components, including hulls (mesocarps and epicarps), seed coats (testas), and kernels (seeds), all contribute to variable aflatoxin content in pistachios. Fresh pistachio kernels were individually inoculated with Aspergillus flavus and incubated 7 or 10 days. Hulled, shelled kernels were either left intact or wounded prior to inoculation. Wounded kernels, with or without the seed coat, were readily colonized by A. flavus and after 10 days of incubation contained 37 times more aflatoxin than similarly treated unwounded kernels. The aflatoxin levels in the individual wounded pistachios were highly variable. Neither fungal colonization nor aflatoxin was detected in intact kernels without seed coats. Intact kernels with seed coats had limited fungal colonization and low aflatoxin concentrations compared with their wounded counterparts. Despite substantial fungal colonization of wounded hulls, aflatoxin was not detected in hulls. Aflatoxin levels were significantly lower in wounded kernels with hulls than in kernels of hulled pistachios. Both the seed coat and a water-soluble extract of hulls suppressed aflatoxin production by A. flavus.

Oxidative damage to DNA induced by areca nut extract.

In Taiwan, people chew betel quid which contains tender areca nut with husk. In other countries, people prefer ripe and dried areca nut without husk. In this study, we compared the reactive oxygen species-induced oxidative DNA damage in isolated DNA and CHO-K1 cells between treatments with tender areca nut extract (ANE) and ripe ANE. Incubation of these two ANE preparations with isolated DNA generated 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in an alkaline environment in a dose-dependent manner. Ripe ANE generated higher levels of 8-OH-dG compared to tender ANE. The addition of iron(II) (100 microM) resulted in 1.4- and 3.1-fold increases of 8-OH-dG when incubated with 1 mg/ml each of tender and ripe ANE. In testing the effect of ANE to cellular DNA, CHO-K1 cells were used for its documented sensitivity to reactive oxygen species. In CHO-K1 cells, ripe ANE was more cytotoxic than tender ANE following an 18-h incubation. The cytotoxicity to CHO-K1 cells was positively correlated with the formation of 8-OH-dG following tender (r=0.97) and ripe (r=0.91) ANE treatment. Addition of the iron chelating agent o-phenanthroline (10 and 20 microM) to cells prior to ri ANE exposure significantly increased (p<0.05) the survival of CHO-K1 cells. In addition, ripe ANE induced dichlorofluorescein-mediated fluorescence which indicated the formation of hydrogen peroxide in CHO-K1 cells. In conclusion, this study demonstrated that ANE-induced oxidative damage to isolated and cellular DNA which may result from the generation of hydrogen peroxide, and iron may serve as a catalyst in this process. Furthermore, ripe ANE generated higher oxidative DNA damage levels compared to tender ANE.

Dose-dependent genotoxic effect of pan masala and areca nut in the Salmonella typhimurium assay.

Aqueous extracts of different brands of pan masala and scented supari were tested for mutagenicity by the Salmonella typhimurium assay using tester strains TA98 and TA100. These extracts were found to be mutagenic to both tester strains. The mutagenic effects of pan masala and scented supari extracts were similar to that produced by areca nut extract. The addition of 500 ppm saccharin to the supari extracts did not alter the mutagenic response.

In vitro genotoxic effects of areca nut extract and arecoline.

The genotoxic potential of the aqueous extract of areca nut as well as arecoline, the major alkaloid of the areca nut, was tested with the help of cytogenetic markers such as sister-chromatid exchanges and chromosome aberrations, utilizing Chinese hamster ovary (CHO) cells. The continuous-treatment and pulse-treatment schedules yielded dose-dependent elevations in the frequencies of sister-chromatid exchange and chromosomal aberration in CHO cells, indicating a genotoxic effect of both the extract and arecoline. The results also imply that, besides arecoline, there may be some other water-extractable substances in the areca nut that make the extract more genotoxic. The chromosome damage was found to be more severe on treating the cells with low concentrations and for longer duration, which mimic the effects of chronic areca nut consumption.

Black walnut (Juglans nigra) toxicosis: a model for equine laminitis.

Twelve light horse geldings developed laminitis within 8 to 12 h of being dosed by nasogastric tube with an aqueous extract of black walnut (Juglans nigra). Four of the 12 horses developed the severe signs of grade 3 laminitis (lame at a walk, refused to lift feet). Laminitis was accompanied by mild depression and limb oedema. There was no evidence of shock or colic. The horses developed neutropenia by 4 h after dosing with the extract, which shifted to a relative neutrophilia by 8 to 12 h. Minimal increases in plasma epinephrine and cortisol concentrations were suggested in severely affected horses. Severe laminitis was characterized by necrosis of dermal tips of dorsal primary epidermal laminae. A proliferative epithelial response in these laminae was distinguished by numerous mitotic figures and clusters of epithelial cells. This evidence suggests that black walnut toxicosis is not only a consistent clinical model, but is also a reliable clinico-pathological and pathological model for study of the pathogenesis and treatment of laminitis.

Toxicity of Abrus precatorius in Nubian goats.

Eight Nubian goats were given Abrus precatorius seed at 2, 1 and 0.5 g/kg/day by stomach tube. Six goats receiving the plant seed at 2 and 1 g/kg died between days 2 and 5. One goat receiving Abrus seed at 0.5 g/kg/day died on day 32 and the other animal in the group was killed on day 33. The main signs of Abrus poisoning were inappetence, bloody diarrhea, dyspnea, dehydration, loss of condition and recumbency. The lesions were fatty change and necrosis of hepatocytes and renal convoluted tubules, pulmonary hemorrhage, edema and emphysema, and erosions of the abomasal and intestinal epithelium. These changes were accompanied by increases in GOT and gamma GT activities and urea, creatinine, sodium and potassium and by decreases in total protein and albumin in the serum of Abrus-poisoned goats. The blood cell changes indicated hemoconcentration.

The toxicity of yellow oleander (Thevetia neriifolia juss) seed kernels to rats.

Toxic effects of yellow oleander (Thevetia neriifolia Juss) seed kernels were evaluated against the roof rat (Rattus rattus Linn). Crushed ground seed kernels were fed with bait at 20 and 30% concentrations. The bait was fed up to mortality or for a maximum of 10 d. Major signs of poisoning observed were hind limb paralysis, rolling of the body on the long axis, circular flailing of the tail, muscular twitch, tetanic convulsions, tremors, collapse and death. Significant reductions in the rats' weights were observed. The observed mortalities were 16/20 and 18/20 with the above respective doses. Statistically significant reductions in hemoglobin, red blood cell count, total leucocyte count and neutrophils, and increased lymphocytes were observed. Reductions in blood glucose and serum proteins, and increased lymphocytes were observed. Reductions in blood glucose and serum proteins, and increased BUN, SGOT and LDH, were also significant. Histopathological studies showed inflammatory and degenerative changes in the liver and kidney. Severe to moderate fatty metamorphosis, congestion, hepatocytolysis, nuclear degeneration, pyknosis, and necrosis were major changes in the liver. Proliferation of glomerular endothelium, hypercellularity of the glomerulus, necrosis of convoluted tubular epithelium, disappearance of nuclei and pyknosis were important changes in the kidney cortical region. Atrophy, erosion and inflammatory changes were observed in the stomach mucosal linings.

Embryotoxicity of betel nuts in mice.

The fetotoxic potential of betel nuts was investigated in Swiss albino mice. Total aqueous extracts of ripe betel nuts of unprocessed and processed varieties were administered to pregnant animals at dose levels of 1, 3 and 5 mg/day/mouse (27 +/- 1 g body wt) through days 6-15 of gestation. The dams were sacrificed prior to term and the fetuses were examined for morphological, visceral and skeletal anomalies. The treatments resulted in increased resorptions as well as dead fetuses. Fetal weight was adversely affected as indicated by the dose related reduction in average body weight of live fetuses. No major morphological, visceral and skeletal defects, apart from hematomas, curved tails and a few incidences of rib anomalies, were observed. There was, however, a dose-related decrease in the number of fetuses possessing ossified coccygeal vertebrae while an increase in the number of fetuses with unossified 5th metacarpals. This indicated a delay in skeletal maturity, particularly in those fetuses exposed prenatally to the betel nut extract of the unprocessed variety.

Liver cell cancer--intervention studies.

The field studies leading to possible intervention procedures are reviewed. Currently the most promising form of intervention is the prevention of aflatoxin contamination of foodstuffs. It is essential that these are monitored and their efficacy in lowering the incidence of liver cancer measured. The association of liver cancer with hepatitis B infection may be a confounding factor and the impact of this on the study population must also be considered. The imminent production of vaccines for hepatitis B infection may provide an alternative or additional mode of intervention. The possibilities for intervention in liver cell cancer appear one of the brighter prospects for primary prevention of a cancer.