Vildagliptin, a dipeptidyl peptidase-4 inhibitor, reduces betel-nut induced carcinogenesis in female mice.

AIM: Betel-nut, a popular masticatory among Southeast Asian populations is a class I carcinogen, previously associated with dyslipidemia and aberrant lipid metabolism, and is reported to be used more frequently by females, than males. This study investigates the potential of repurposing the anti-diabetic drug, vildagliptin, a dipeptidyl peptidase-4 inhibitor, for alleviating the oncogenic condition in female Swiss Albino mice administered an aqueous extract of betel-nut (AEBN) orally (2 mg ml-1) for 24 weeks. MAIN METHODS: Tissues were investigated by histopathological, immunohistochemical and apoptosis assays. Biochemical analyses of oxidative stress markers and lipid profile were performed using different tissues and sera. The expressions of different proteins involved in lipid metabolism and oncogenic pathways were evaluated by Western blotting. KEY FINDINGS: AEBN induced carcinogenesis primarily in the liver by significantly impairing AMPK signaling, inducing oxidative stress, activating Akt/mTOR signaling, increasing Ki-67 immunoreactivity and cyclin D1 expression, and significantly diminishing apoptosis. Co-administration of AEBN with vildagliptin (10 mg kg-1 body weight) for 8 weeks reduced liver dysplasia, and significantly decreased free palmitic acid, increased free oleic acid, normalized lipid profile, decreased oxidative stress, cyclin D1 expression, Ki-67 immunoreactivity, and Bcl2 expression, and increased the ratio of apoptotic/non-apoptotic cells. Mechanistically, vildagliptin elicited these physiological and molecular alterations by restoring normal AMPK signaling and reducing the cellular expressions of FASN and HMGCR, restoring AMPK-dependent phosphorylation of p53 at Ser-15 and reducing Akt/mTOR signaling. SIGNIFICANCE: These results indicate that vildagliptin may alleviate betel-nut induced carcinogenesis in the liver of female mice.

Evaluation of 13-week repeated oral dose toxicity of Areca catechu in F344/N rats.

This study investigated the potential toxicity of the Areca catechu water extract after 13-week repeated oral administration at 0, 166.7, 500, and 1500 mg/kg/day in rats. During the study period, clinical signs, mortality, body weight, food consumption, water consumption, urinalysis, estrous cycle, sperm count and motility, ophthalmoscopy, hematology, serum biochemistry, gross pathology, organ weights, and histopathology were examined. At 1500 mg/kg/day, both sexes exhibited an increase in the incidence of abnormal clinical signs, which included, decreased body weight gain and food consumption, and increased urine bilirubin, ketone bodies, specific gravity, and protein and kidney weight. An increase in liver weight and estrous cycle alterations was observed in females. Serum biochemical and histopathological investigations revealed an increase in the levels of serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, and the incidence of hepatic necrosis in females. At 500 mg/kg/day, an increase in the incidence of abnormal clinical signs including diarrhea and soiled perineal region, was observed in both sexes. No treatmentrelated effects were observed at 166.7 mg/kg/day. Under the present experimental conditions, the target organs were determined to be the liver, kidney, and female reproductive system in rats. The no-observedeffect level was considered to be 166.7 mg/kg/day in rats.

Low dietary n-6/n-3 polyunsaturated fatty acid ratio prevents induced oral carcinoma in a hamster pouch model.

Dietary fatty acid patterns have been linked to the prevalence of certain cancers, however in oral carcinoma is limited. Thus, we investigated the chemopreventive effects of various dietary n-6 and n-3 fatty acids in a 9,10-dimethyl-1,2-benz[a]-anthracene (DMBA)- and betel quid extract (BQE) -induced hamster oral cancer model. Thirty 6-week-old adult male hamsters were housed and divided into normal, low, and high dietary n-6 and n-3 fatty acid groups under DMBA + BQE treatment for 16 weeks. The right buccal pouch of all hamsters were evaluated by tumor number, volume, burden and selected inflammatory parameters. The results indicate that the low dietary n-6/n-3 fatty acid group exhibited a significantly lower tumor number, volume, and burden than those of the other groups. Furthermore, this group had significantly lower nuclear factor-κB, proliferating cell nuclear antigen, and cyclin D1 expression in the right buccal pouch tissue. In conclusion, the lower dietary n-6/n-3 fatty acid ratio exerted chemopreventive effects in the DMBA- and BQE-induced hamster oral cancer model.

[Systematic evaluation for safety of traditional Chinese medicine Areca catechu and its preparations].

The authors systemically evaluated and analyzed the safety of Areca catechu from domestic and foreign literatures about the herbal origin, toxicity recorded in ancient/current documents, safety case reports of clinical A. catechu, experimental studies on toxicity in recent years, and differences of safety risk between edible and medicinal A. catechu. Subsequently, they proposed a preliminary summary about the clinical characteristics and potential risk factors of safety related cases of A. catechu and its preparations. According to the authors, although clinical adverse events of A. catechu were fewer and controllable, clinicians shall stillstrictly standardize its application, and rationally combine it with other herbs, while strengthening fundamental and clinical studies related to safety, so as to give better guidance to safety application of A. catechu in clinic.

Using the "target constituent removal combined with bioactivity assay" strategy to investigate the optimum arecoline content in charred areca nut.

Charred areca nut (CAN) is used to treat dyspepsia and abdominal distension in children. However, reports revealed that arecoline, the most important active constituent of CAN, possesses potential toxicities. This study was designed to investigate the optimum arecoline content in CAN, using the "target constituent removal combined with bioactivity assay" strategy. Based on PTLC method, we prepared CAN lacking all arecoline (WAC-100R) and a series of different ratios of arecoline-removed CAN samples (WAC-Rx). MTT and acute toxicity assays indicated that decreasing content by 50% decreased CAN toxicity significantly. Animal results revealed arecoline contents over 50% could guarantee the beneficial effects of CAN on gastrointestinal tract. Additionally, decreasing arecoline content in CAN by 50% decreased its pro-apoptotic effects significantly. Furthermore, decreasing 50% arecoline content in CAN down-regulated the expressions of Cleaved-Caspase-3, c-jun, c-fos, COX-2, PGE2, and IL-1α. Thus, our results revealed that CAN with 50% arecoline content (WAC-50R) has similar beneficial effects on the gastrointestinal tract to CAN, whereas its toxicity was decreased significantly. Collectively, our study suggested that the strategy of "target constituent removal combined with bioactivity assay" is a promising method to identify the optimum arecoline content in CAN, which is approximately 0.12%.

Proteomics Analysis Reveals Involvement of Krt17 in Areca Nut-Induced Oral Carcinogenesis.

The areca nut is a known carcinogen that causes oral cancer in individuals in Southeast Asia, but the molecular mechanism that leads to this malignancy is still unclear. To mimic the habit of areca nut chewing, our laboratory has established four oral cancer cell sublines (SAS, OECM1, K2, C9), which have been chronically exposed to areca nut extract (ANE). To elucidate the molecular basis of areca nut-induced oral carcinogenesis, the differential proteomes between oral cancer cells and the ANE-treated sublines were determined using isobaric mass tag (iTRAQ) labeling and multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Over 1000 proteins were identified in four sublines, and 196 proteins were found to be differentially expressed in at least two ANE-treated sublines. A bioinformatic analysis revealed that these proteins participate in several pathways, and one of the most prominent pathways was the regulation of epithelial to mesenchymal transition (EMT). In all, 24 proteins including Krt17 were confirmed to be differentially expressed in the ANE-treated sublines. To reveal additional information on the mechanism of ANE-induced carcinogenesis, Krt17 was further investigated. Krt17 knockdown significantly suppressed ANE-induced cell migration and invasion and modulated the EMT process. Furthermore, in a murine model of carcinogen-induced (arecoline cocktail, an active compound of ANE) oral cancer, Krt17 was significantly up-regulated in all hyperplastic tissues and in carcinoma tissues (p < 0.001). In conclusion, we have identified a proteome of oral cancer cells that is associated with chronic areca nut exposure. Krt17 was demonstrated to contribute to areca nut-induced oral malignancy. The results of this study contribute to risk assessment, disease prevention and other clinical applications associated with areca nut-induced oral cancer.

Areca nut components stimulate ADAM17, IL-1α, PGE2 and 8-isoprostane production in oral keratinocyte: role of reactive oxygen species, EGF and JAK signaling.

Betel quid (BQ) chewing is an etiologic factor of oral submucous fibrosis (OSF) and oral cancer. There are 600 million BQ chewers worldwide. The mechanisms for the toxic and inflammatory responses of BQ are unclear. In this study, both areca nut (AN) extract (ANE) and arecoline stimulated epidermal growth factor (EGF) and interleukin-1α (IL-1α) production of gingival keratinocytes (GKs), whereas only ANE can stimulate a disintegrin and metalloproteinase 17 (ADAM17), prostaglandin E2 (PGE2) and 8-isoprostane production. ANE-induced EGF production was inhibited by catalase. Addition of anti-EGF neutralizing antibody attenuated ANE-induced cyclooxygenase-2 (COX-2), mature ADAM9 expression and PGE2 and 8-isoprostane production. ANE-induced IL-1α production was inhibited by catalase, anti-EGF antibody, PD153035 (EGF receptor antagonist) and U0126 (MEK inhibitor) but not by α-naphthoflavone (cytochrome p450-1A1 inhibitor). ANE-induced ADAM17 production was inhibited by pp2 (Src inhibitor), U0126, α-naphthoflavone and aspirin. AG490 (JAK inhibitor) prevented ANE-stimulated ADAM17, IL-1α, PGE2 production, COX-2 expression, ADAM9 maturation, and the ANE-induced decline in keratin 5 and 14, but showed little effect on cdc2 expression and EGF production. Moreover, ANE-induced 8-isoprostane production by GKs was inhibited by catalase, anti-EGF antibody, AG490, pp2, U0126, α-naphthoflavone, Zinc protoporphyrin (ZnPP) and aspirin. These results indicate that AN components may involve in BQ-induced oral cancer by induction of reactive oxygen species, EGF/EGFR, IL-1α, ADAMs, JAK, Src, MEK/ERK, CYP1A1, and COX signaling pathways, and the aberration of cell cycle and differentiation. Various blockers against ROS, EGF, IL-1α, ADAM, JAK, Src, MEK, CYP1A1, and COX can be used for prevention or treatment of BQ chewing-related diseases.

Changes in buccal micronucleus cytome parameters associated with smokeless tobacco and pesticide exposure among female tea garden workers of Assam, India.

Assam is the highest tea producing state in India. A large number of workers are engaged in various units of tea industry. There are few reports on the health status of the tea garden workers. The present cytogenetic biomonitoring study was undertaken to investigate the genotoxic effect associated with workers in tea industries in southern Assam. Smokeless tobacco chewing along with betel nut is very common practice among the workers. Workers also get exposed periodically to mixture of pesticides. Employing buccal micronucleus cytome assay, exfoliated buccal cells were analyzed in 90 female tea garden and compared to 90 age and sex matched non-chewer control as well as 70 chewers who are not tea garden workers. Statistically significant (p<0.001) increase in genotoxic and cell death parameters was observed in tea garden workers compared to both the control groups. The frequency of cell proliferation biomarkers was highest in the chewer controls whereas genotoxic and cell death parameters were highest in tea garden workers. Linear correlation analysis revealed strong positive correlation between the duration of occupation and the frequency of micronucleus (r=0.597; p<0.001) as well as cell death parameters (r=0.588; p<0.001). Amount of chewing also had significant positive correlation with micronucleus frequency (r=0.243 or 5.9%; p<0.05) and cell death parameters (r=0.217; p<0.05). A statistically significant decrease in total RBC count, haemoglobin content as well as acetylcholine esterase in the blood of exposed individuals was observed. The average BMI among the tea garden workers was relatively lower compared to the control group. Pesticide exposure and chewing areca nut along with smokeless tobacco use may be responsible for changes in cytome parameters in exfoliated buccal cells.

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.

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.

Altered BRCA1 and BRCA2 responses and mutation of BRCA1 gene in mice exposed chronically and transgenerationally to aqueous extract of betel nut (AEBN).

The Brca1 and Brca2 tumor suppressor genes are involved in the maintenance of genomic integrity as they facilitate error free DNA repair. This study was designed to understand the role of Brca1 and Brca2 in betel nut (BN) induced chronic and transgenerational carcinogenesis in mice. Young male and female Swiss Albino mice were chronically as well as transgenerationally exposed to aqueous extract of betel nut (AEBN) in drinking water (2 mg ml(-1)) for up to 24 weeks. In chronically exposed mice, the levels of Brca1 and Brca2 proteins were elevated to approximately 1.4-fold over the age matched controls after 2 weeks of exposure to AEBN, followed by a decline below the controls. In transgenerationally exposed mice, both Brca1 and Brca2 proteins remained below the controls from the onset of AEBN exposure and rapidly declined further, indicating a loss of tumor suppressor protection. Nucleotide sequencing of exon 11 of Brca1 and exon 27 of Brca2 did not reveal mutation in liver nodules of chronically exposed mice, while a G → C mutation Brca1 was observed in liver nodules as well as in solid tumors developing in transgenerationally exposed mice. Thus, the genomic instability arising due to the lowering in the levels of Brca1 and Brca2 proteins and mutation in exon 11 of Brca1 gene contributed to the increased risk of cancer in mice exposed transgenerationally to AEBN.

Reproductive toxic potential of panmasala in male Swiss albino mice.

Some ingredients of panmasala have the ability to penetrate the blood-testis barrier but the reproductive toxic potential of panmasala has not been studied. This study is aimed to assess the possible damage caused by panmasala to male reproductive system in mice. Swiss albino male mice were randomly divided into 7 groups receiving either standard control diet or panmasala-containing diet. Three doses (0.5%, 1.5% and 3%) of panmasala plain (PMP) as well as panmasala with tobacco (PMT)-gutkha were given for a period of 6 months. Assessment of organ weight, sperm count and morphology, spermatid count, sperm production, testicular 17ß-hydroxysteroid dehydrogenase (17ß-HSD) activity and histology were conducted. A nonsignificant decrease in absolute and relative weight of testis and epididymis was observed. Spermatid count, sperm count and production were significantly decreased and 17ß-HSD activity was found considerably declined at 3% of both PMP- and PMT-treated groups as compared to control. The histological observations revealed panmasala induced testicular damage. Abnormal morphology of sperm head shape was significantly elevated in higher doses of both types of panmasala-treated groups than control. The results suggests that panmasala has reproductive toxic potential and more alteration is seen with gutkha as compared to panmasala plain, indicating that similar effects might also be possible in humans.

Potential analgesic, anti-inflammatory and antioxidant activities of hydroalcoholic extract of Areca catechu L. nut.

The hydroalcoholic extract of Areca catechu L. (ANE) nut was screened for its analgesic, anti-inflammatory and in vitro antioxidant potential. Three doses of ANE (250, 500 and 1000 mg/kg orally) were tested for analgesic and anti-inflammatory activities. Evaluation of analgesic activity of ANE was performed using hot plate and formalin test in mice. ANE showed maximum increase in hot plate reaction time (56.27%, p<0.01), while reduced the duration of licking/biting behaviors in first (39.45%, p<0.05) and second (92.71%, p<0.01) phases of the formalin test indicating significant analgesic activity. ANE reduced the paw edema considerably (86.79% inhibition after 24h, p<0.01) in dose-dependent manner compared to carrageenan-induced rat. In addition, in vitro antioxidant activity of ANE was investigated by total phenolic content (TPC) and hydrogen peroxide assay. The IC(50) observed in hydrogen peroxide assay was 83.14 µg/ml and TPC 120.56±21.09 mg QE/g. Altogether, these results suggest that the hydroalcoholic extract of Areca catechu could be considered as a potential analgesic, anti-inflammatory and antioxidant agent.

Is the blood donated by habitual nut quid chewers suitable for use in transfusion?

BACKGROUND/PURPOSE: Betel quid (BQ) chewing is a popular oral masticatory activity, and there are approximately 600 million BQ chewers worldwide. Although chewing BQ has been linked to the patho-genesis of oral cancer, leukoplakia, and oral submucous fibrosis. The question whether the mixed constituents present in areca nut, which may exert cytotoxic effects on red blood cells (RBCs), has never been addressed. METHODS: Heparinized blood specimens were obtained with informed consent from healthy laboratory personnel. RBCs were separated with the standard procedure and adjusted to 10% hematocrit with PBS. Various concentrations of areca nut extract (ANE; 100-800 microg/mL) were added to these RBC preparations and incubated at 37 degrees C for 4 hours. Two portions (0.4 mL each) of the incubated RBCs were then used for measuring osmotic deformability index and for observing RBC morphology with scanning electron microscopy. The remaining RBCs were used for determining membrane sulfhydryl groups and protein profiles by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Blood incubated with various concentrations of ANE showed concentration-dependent decreases in osmotic deformability index and membrane sulfhydryl groups. Membrane protein profiles revealed a significant loss of the band 3 fraction, with the concomitant appearance of several new protein bands in the electropheretogram. Finally, drastic morphological changes of ANE-treated RBCs were observed. CONCLUSION: We suggest that to assure the quality of transfusion, the blood donated by a habitual BQ chewer should be used with caution because of its possible contamination with areca nut ingredients that may be cytotoxic to RBCs.

A characterization of the antioxidant enzyme activity and reproductive toxicity in male rats following sub-chronic exposure to areca nut extracts.

In the present study, areca nut extracts (ANE) administered to male rats by gavage at a dose of 100mg/kg/day for a period of 15, 30, or 45 days resulted in signs of reproductive toxicity. ANE administration resulted in a significant decline (30-57% in epididymal sperm count and 27-61% in sperm motility) as well as substantial abnormalities in sperm morphology. Significant variances in activities of antioxidant enzymes were also observed. Malondialdehyde (MDA) levels, which represent the level of lipid peroxidation, increased by 16-188% and levels of sialic acid decreased by 2-46% compared with that in controls. These results indicate that ANE induced spermatogenic damage, as indicated by a decrease in sperm counts and sperm motility as well as the activity of antioxidant enzymes, an increase in sperm abnormalities, and alterations in sialic acid and MDA levels. Such effects reflect that ANE administration resulted in reactive oxygen species (ROS)-induced oxidative stress in the testis, cauda epididymis, and sperm of male rats.

Asb6 upregulation by Areca nut extracts is associated with betel quid-induced oral carcinogenesis.

Betel quit (BQ) chewing is a popular habit, especially in southern and southeastern Asia. Areca nut extracts (ANE), the major components of BQ, have been documented to induce reactive oxygen species, and consequently to cause genetic damage. ANE usage is tightly linked to oral cancer; however, the details of the molecular mechanism that results in carcinogenesis remain unclear. Previously, we successfully established HaCaT cells surviving from the long-term exposure of sublethal doses of ANE (Lai KC, Lee TC. Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts. Mutat Res 2006;599:66-75). Here, we identified the upregulation of Asb6, a coupling protein to the APS adapter protein, which is involved in insulin signaling for glucose transportation, of normal keratinocytes and oral cancer cells under ANE treatment. Immunohistochemical analyses of Asb6 on oral squamous cell carcinoma (OSCC) tissues (n=57) demonstrated the positive correlation between Asb6 upregulation (cancerous tissues versus adjacent normal tissues) and clinicopathological features. We showed that the combination of ANE-enhanced Asb6 expression in vitro and Asb6 upregulation in OSCC patients leads to poor survival status. In conclusion, our results suggest that upregulated Asb6 could act as a prognostic marker for oral cancer.

Up-regulation of inflammatory signalings by areca nut extract and role of cyclooxygenase-2 -1195G>a polymorphism reveal risk of oral cancer.

Because the mRNA expression of cyclooxygenase-2 (COX-2) is up-regulated by arecoline in human gingival fibroblasts, as shown in our previous study, we further investigated the mRNA expression level of COX-2 and its upstream effectors in three oral epithelial carcinoma cell lines (KB, SAS, and Ca9-22) by using areca nut extract (ANE) and saliva-reacted ANE (sANE). A case-control study of 377 oral squamous cell carcinoma (OSCC) patients and 442 controls was conducted to evaluate the gene-environment interaction between COX-2 promoter polymorphisms and substance use of alcohol, betel quid, and cigarettes (ABC) in risk of OSCC. The heterogeneous characteristics of the oral site and the COX-2 -1195G>A polymorphism in these cell lines showed diverse inflammatory response (KB>>Ca9-22>SAS) after 24-hour ANE/sANE treatments, and the COX-2 up-regulation might be mostly elicited from alternative nuclear factor-kappaB activation. In the case-control study, betel chewing [adjusted odds ratios (aOR), 42.2] posed a much higher risk of OSCC than alcohol drinking and cigarette smoking (aORs, 2.4 and 1.8, respectively), whereas the COX-2 -1195A/A homozygote presented a potential genetic risk (OR, 1.55). The strongest joint effect for OSCC was seen in betel chewers with -1195A/A homozygote (aOR, 79.44). In the non-betel chewing group, the -1195A/G and A/A genotypes together with the combined use of alcohol and cigarettes increased risk to 15.1-fold and 32.1-fold, respectively, compared with the G/G genotype without substance use. Taken together, these findings illustrate a valuable insight into the potential role of the COX-2 promoter region in contributing to the development of betel-related OSCC, including ANE/sANE-induced transcriptional effects and enhanced joint effects of COX-2 -1195A allele with substance use of ABC.

Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts.

Chewing betel quid (BQ) is a popular habit worldwide. A causal association between BQ chewing and oral cancer has been well documented. Emerging evidence indicates that sustained exposure to stress induces epigenetic reprogramming of some mammalian cells and increases the mutation rate to accelerate adaptation to stressful environments. In this study, we first confirmed that 24-h treatment with areca nut extracts (ANE; a major component of BQ) at doses over 40 microg/ml induced mutations at the hypoxanthine phosphoribisyltransferase (HPRT) locus in human keratinocytes (HaCaT cells). We then investigated whether the stress of long-term exposure to sublethal doses of ANE (0, 5 and 20 microg/ml for 35 passages) could enhance genetic damage to HaCaT cells. Compared to cells exposed to 0 or 5 microg/ml ANE, cells exposed to 20 microg/ml ANE were slightly but significantly more resistant to a 72-h treatment with ANE and its major ingredients, arecoline and arecaidine, but did not develop cross-resistance to other BQ ingredients or alcohol. The cells that received 20 microg/ml ANE for 35 passages also had a significantly increased mutation frequency at the HPRT locus and an increased frequency in the appearance of micronuclei compared to lower doses. Moreover, increased intracellular levels of reactive oxygen species and 8-hydroxyguanosine in cells exposed to 20 microg/ml ANE suggested that long-term ANE exposure results in the accumulation of oxidative damage. However, cells subjected to long-term treatment of 20 microg/ml ANE contained higher levels of glutathione than unexposed cells. Therefore, after long-term exposure to sublethal doses of ANE, intracellular antioxidative activity may also be enhanced in response to increased oxidative stress. These results suggest that stress caused by long-term ANE exposure enhances oxidative stress and genetic damage in human keratinocytes.