Areca nut‐induced micronuclei and cytokinesis failure in Chinese hamster ovary cells is related to reactive oxygen species production and actin filament deregulation

Epidemiological studies have shown a strong association between environmental exposure to betel quid (BQ) and oral cancer. Areca nut (AN), an ingredient of BQ, contains genotoxic and mutagenic compounds. In this study, we found that AN extract (ANE) inhibited the growth of Chinese hamster ovary cells (CHO‐K1) in a dose‐ and time‐dependent manner. Intracellular reactive oxygen species (ROS) levels and micronuclei (MN) frequency were significantly increased following ANE treatment in CHO‐K1 cells. Addition of catalase markedly inhibited ANE‐induced MN formation, indicating that ANE‐induced genotoxicity was correlated with intracellular H₂O₂. Incubation of CHO‐K1 cells with ANE (400–800 μg/ml) for 24 hr caused G2/M arrest, and prolonged exposure to ANE (800 μg/ml) significantly induced cell death. Surprisingly, ANE itself caused cytokinesis failure and subsequent increase in binucleated cell formation. Coexposure to catalase (2,000 U/ml) and ANE (800 μg/ml) reduced the generation of binucleated cells, indicating that ANE‐induced cytokinesis failure was associated with oxidative stress. Following prolonged exposure to ANE, an accumulation of hyperploid/aneuploid cells concomitant with bi‐, micro‐ or multinucleated cells was found. In summary, our results demonstrate that ANE exposure to CHO‐K1 cells caused increased MN frequency, G2/M arrest, cytokinesis failure, and an accumulation of hyperploid/aneuploid cells. These events are associated with an increase in intracellular H₂O₂ level and actin filament disorganization. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

L‐type Ca2+ channel opener BayK 8644‐induced Ca2+ influx and Ca2+ release in human oral cancer cells (OC2)

The effect of BayK 8644, a chemical widely used to activate L‐type Ca²⁺ channels, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human oral cancer cells (OC2) has not been explored to date. The present study examined whether BayK 8644 altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura‐2. BayK 8644 (10 pM–10 µM) increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. BayK 8644‐induced Ca²⁺ influx was blocked by nifedipine, but was not altered by the store‐operated Ca²⁺ entry inhibitors, econazole and SKF96365; protein kinase C modulators phorbol 12‐myristate 13‐acetate (PMA) and GF109203X; the protein kinase A inhibitor H89; and the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺‐free medium, after pretreatment with 1 µM BayK 8644, 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; and conversely, thapsigargin pretreatment abolished BayK 8644‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change BayK 8644‐induced [Ca²⁺]_i rises. Collectively, in OC2 cells, BayK 8644 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum; and Ca²⁺ influx via L‐type Ca²⁺ channels. Drug Dev Res 69: 2008. © 2008 Wiley‐Liss, Inc.     

Additive effect modification of hepatitis B surface antigen and e antigen on the development of hepatocellular carcinoma.

To assess the role of hepatitis B e antigen (HBeAg) and its interaction with hepatitis B surface antigen (HBsAg) on the development of hepatocellular carcinoma (HCC), this case-control study included 361 age- and sex-matched pairs of patients with histologically proven HCC and healthy control subjects. HBsAg, HBeAg and antibody to HBeAg (anti-HBe) were detected by radioimmunoassay. Antibodies to hepatitis C virus (anti-HCV) were detected by second-generation enzyme immunoassay. The prevalences of HBeAg (20.2%), HBsAg (80.3%) and anti-HCV (29.5%) in cases were higher than in controls (1.9%, 20.7%, and 2.7% respectively; each P < 0.0001). Using patients negative for HBsAg, HBeAg and anti-HBe as a referent group, univariate analysis indicated that HBsAg alone or HBsAg and HBeAg were risk factors for HCC (P for trend < 0.0001). Calculation of incremental odds ratio indicated that there was additive interaction between HBsAg and HBeAg. Multivariate analysis indicated that HCC development was strongly associated with the presence of HBeAg (odds ratio, 8.1; 95% confidence interval, 2.4-27.1), HBsAg (odds ratio, 68.4; 95% confidence interval, 20.5-227.8) and anti-HCV (odds ratio, 59.3; 95% confidence interval, 13.6-258.4). In conclusion, HBsAg, HBeAg and anti-HCV are independent risk factors for HCC. There is additive and independent effect modification between HBsAg and HBeAg on the development of HCC.     

Pedunculated Hepatocellular Carcinoma: Clinicopathologic Study of 18 Surgically Resected Cases

We present the clinical features and outcomes of 18 surgically treated pedunculated hepatocellular carcinomas (P-HCCs). Hepatocellular carcinoma is a notorious, hyperendemic disease in Taiwan. Pedunculated HCC, although not a novel finding, has been recognized and diagnosed early by various imaging modalities. However, the clinicopathologic picture has not been fully clarified, and the prognosis varies in each report. From 1986 to 1998 the clinical features of 18 surgically treated cases of P-HCC were reviewed, including demographics, laboratory data, operative findings, pathologic features, and follow-up results. Factors that may influence the outcomes were also analyzed. Clinical features and outcomes of 414 patients with nonpedunculated hepatocellular (HCC) were summarized for comparison. Of 432 surgical resected hepatocellular carcinomas, 18 (4.2%) were P-HCCs. Larger tumor size, more capsule formation, less vascular invasion, and wider resection margins were significantly prominent in the patients in P-HCC group compared with those in the NP-HCC group. Multivariate stepwise logistic regression analysis revealed that the P-HCC group had significantly larger tumors and wider resection margins. The 1-, 3-, and 5-year survival rates of P-HCC patients were 88.3%, 77.4%, and 45.6%, respectively. A significant difference in survival was found between the P-HCC and NP-HCC groups. P-HCC patients without vascular invasion might have a significantly better survival demonstrated by log-rank analysis stratified by capsular invasion, vascular invasion, resection, and tumor size. We present the clinical features and outcomes of 18 surgically treated pedunculated HCCs. Pedunculated HCCs might have a better survival than conventional HCCs after hepatic resection, especially if there is no vascular invasion.     

Hydrogen peroxide-induced intracellular acidosis and electromechanical inhibition in the diseased human ventricular myocardium

Accumulation of oxygen free radicals is an important mediator of post-ischemia/reperfusion cardiac dysfunction. However, oxidative injury has not been well characterized in human cardiac tissues. In the present study, we superfused hydrogen peroxide (H(2)O(2)) into the diseased human ventricle in order to assess the effects of oxygen free radicals on the electromechanical parameters and the intracellular pH (pH(i)), and to test the ability of certain potential cardioprotective agents, including scavengers of hydrogen peroxide (dibenzamidostilbene disulfonic acid; DBDS), the.OH free radical (N-(mercaptopropionyl)-glycine; N-MPG), and the HOCl free radical (L-methionine), to protect against oxidative injury. Disease human ventricular tissues were obtained from patients undergoing heart transplantation. Electrophysiological experiments were performed using a traditional micropipette, while the pH(i) was measured by microspectrofluorimetry. We found that (a) H(2)O(2) (30 microM-3 mM) induced a significant dose-dependent intracellular acidosis, (b) H(2)O(2) (30 microM-3 mM) had a notable dose-dependent biphasic effect on the contractile force (an increase, followed by a decrease), while moderate concentrations of H(2)O(2) also inhibited the generation of action potential and increased the diastolic resting force significantly, and (c) N-MPG caused significant block of both the intracellular acidosis and the electromechanical inhibition induced by 3 mM H(2)O(2), whereas L-methionine and DBDS did not. Our data suggest that the toxic effects of H(2)O(2) are caused mainly through the generation of.OH, which is attributed to the intracellular acidosis seen in the diseased human ventricle.     

Spontaneous tumour rupture and prognosis in patients with hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is a common disease in Taiwan. Ruptured HCC is an uncommon and potentially fatal complication of the condition. Information on the impact of ruptured HCC on hepatic resection is, however, limited. METHODS: The clinical features of 60 patients with ruptured HCC who underwent hepatic resection from 1986 to 1998 were reviewed. Clinical features and factors influencing the outcome of 475 patients with non-ruptured HCC were used for comparison. RESULTS: Of 535 surgically resected HCCs, 60 (11.2 per cent) were ruptured. Univariate analysis showed that sudden onset of abdominal pain, physical signs of haemodynamic unstability, reduced haemoglobin level and a raised aspartate aminotransferase level were more frequently found in patients with ruptured HCC than in those with non-ruptured tumours. Multivariate stepwise logistic regression analysis revealed sudden-onset abdominal pain to be the only independently significant factor in patients in the ruptured HCC group. The 1-, 3- and 5-year survival rates of patients with non-ruptured HCC were 72.1, 47.3 and 33.9 per cent, and those of patients with ruptured HCC were 54.2, 35.0 and 21.2 per cent respectively. Similar overall survival rates were found in patients with ruptured and non-ruptured HCC, although patients in the non-ruptured HCC group had a significantly better disease-free survival rate (P = 0.023). CONCLUSION: The presence of sudden-onset abdominal pain is the only independent indicator of ruptured HCC. Hepatic resection, when feasible, is the treatment of choice and can result in an overall survival rate comparable to that of patients with non-ruptured HCC.