Fung-Jou Lu

An ultra-weak chemiluminescence study on oxidative stress in rabbits following acute thermal injury

It is not easy to detect oxygen free radicals directly because of their very short half-life. In the present study, a sensitive ultra-weak chemiluminescence detector was used to detect the generation of oxygen free radicals following thermal injury. Twelve New Zealand white rabbits were used in this study. After anesthesia, the bilateral hind-limbs were exposed to 100 degrees C water for 30 s. Six control animals were exposed to 22 degrees C water to act as a control. The chemiluminescence of whole blood and visceral organs were measured with both luminol-amplified t-butyl hydroperoxide-initiated and lucigenin-initiated methods. The results showed that chemiluminescence of blood was affected significantly by acute thermal injury. The chemiluminescence of blood increased significantly at 1 h following acute thermal injury, reached a peak at 2 h, then decreased but still remained above the control level at 4 h following thermal injury. The results for TBHP-initiated chemiluminescence from visceral organs following acute thermal injury were much higher than that of the control rabbits. The effects of lucigenin-initiated tissue chemiluminescence following acute thermal injury were not statistically significant. It is suggested that the decreased vascular antioxidant activity following local thermal injury is partially contributed by the superoxide pathway; while, the remote pathophysiologic events are mediated by the defective scavenging defenses.

Enhanced oxidative stress and accelerated cellular senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts

Glucose-6-phosphate dehydrogenase (G6PD) is involved in the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the maintenance of the cellular redox balance. The biological effects of G6PD deficiency in nucleated cells were studied using G6PD-deficient human foreskin fibroblasts (HFF). In contrast to that of normal HFF, the doubling time of G6PD-deficient cells increased readily from population doubling level (PDL) 15 to 63. This was accompanied by a significant increase in the percentage of G(1) cells. The slow-down in growth preceded an early entry of these cells into a nondividing state reminiscent of cellular senescence. These cells exhibited a significant increase in level of senescence-associated beta-galactosidase (SA-beta-gal) staining. The importance of G6PD activity in cell growth was corroborated by the finding that ectopic expression of active G6PD in the deficient cells prevented their growth retardation and early onset of senescence. Mechanistically, the enhanced fluorescence in dichlorofluorescin (H(2)DCF)-stained G6PD-deficient cells suggests the possible involvement of reactive oxygen species in senescence. Taken together, our results show that G6PD deficiency predisposes human fibroblasts to retarded growth and accelerated cellular senescence. Moreover, G6PD-deficient HFF provides a useful model system for delineating the effects of redox alterations on cellular processes.

Free radical-scavenging properties of lignin

Lignin is one of the major components of dietary fiber. It is a complex hydrophobic molecule that typically occurs in cell walls with heteroxylans. Our experimental data show that lignin is a free radical scavenger. When the NADH-phenazine methosulfate-nitro blue tetrazolium free radical-producing system is used, an alkali-lignin concentration of 46.29 micrograms/ml that causes 50% inhibition of uric acid production by xanthine oxidase (IC50) is a scavenger of superoxide anion radicals. Spectrophotometric assay has shown that alkali-lignin with an IC50 of 59.08 micrograms/ml inhibits the activity of xanthine oxidase, one of the enzymes related to the production of superoxide anion radicals, and presents a mixed-type noncompetitive inhibition pattern. Using the deoxyribose method, we have found that alkali-lignin is a hydroxyl radical scavenger with an IC50 of 250 micrograms/ml, and using the thiobarbituric acid method, we can see that alkali-lignin inhibits nonenzymatic and enzymatic lipid peroxidation with an IC50 of 72 and 100 micrograms/ml, respectively. Alkali-lignin also hinders the activity of glucose-6-phosphate dehydrogenase, another enzyme related to the generation of superoxide anion radicals, with an IC50 of 123.6 micrograms/ml, and obstructs the growth and viability of cancer (HeLa) cells in a dose-dependent manner. Our experimental results suggest another mechanism whereby the free radical-scavenging activity of lignin in dietary fiber may be involved in the fiber-colon cancer interaction. We also suggest that the ability of dietary fiber to protect against colon cancer may be partly determined by the amount of lignin in dietary fiber as well as the free radical-scavenging ability of lignin.

Loss of heterozygosity at chromosome 13q in hepatocellular carcinoma: identification of three independent regions

Loss of heterozygosity (LOH) on chromosome 13q is one of the most common genetic alterations in hepatocellular carcinoma (HCC) and might be involved in liver cancer development through inactivation of tumour suppressor genes. In order to narrow down the region of 13q loss, we examined the pattern of loss of heterozygosity (LOH) in tumours from 88 HCC patients, using 18 microsatellite markers on 13q. Thirty-eight of the 88 tumours (43%) showed LOH for at least one marker. Of these, two tumours (5%) showed 13q whole arm allelic loss, while the remaining 36 tumours (95%) had partial allelic loss. The LOH pattern defined by the 36 tumours suggested the existence of at least three different smallest common deleted regions which might be involved in the carcinogenesis of HCC. The first, the most centromeric in the 13q12.3 is, close to the BRCA2 gene, defined by D13S171; the second, the most telomeric region in the 13q31-32 band, is defined by D13S154 and D13S157; the third, the intermediate region at 13q14.3, which is near the RB gene, is defined by loci D13S268. The rate of LOH at 13q31-32 was significantly higher in Hepatitis B-surface antigen (HBsAg)-positive patients than HBsAg-negative HCC patients, pointing to a candidate gene related to the development of HBsAg-positive HCCs.

Protective effects of seabuckthorn (Hippophae rhamnoides L.) seed oil against carbon tetrachloride-induced hepatotoxicity in mice.

The present study examined the protective effects of seabuckthorn (Hippophae rhamnoides L., SBT) seed oil on carbon tetrachloride (CCl(4))-induced hepatic damage in male ICR mice. Our results showed that oral administration of SBT seed oil at doses of 0.26, 1.30, and 2.60 mg/kg for 8 weeks significantly reduced the elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride (TG), and cholesterol at least 13% in serum, and the level of malondialdehyde (MDA) in liver at least 22%, that was induced by CCl(4) (1 mL/kg) in mice. Moreover, the treatment of SBT seed oil was also found to significantly increase the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd), and GSH content in liver up to 134%. Our study found that the optimal dose of SBT seed oil was 0.26 mg/kg, as the minimum amount exhibiting the greatest hepatoprotective effects on CCl(4)-induced liver injury. Overall, the hepatoprotective effect of SBT seed oil at all tested doses was found to be comparable to that of silymarin (200 mg/kg) and have been supported by the evaluation of the liver histopathology in mice.

Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide

BackgroundArsenic trioxide (As2O3) exhibits promising anticarcinogenic activity in acute promyelocytic leukemic patients and induces apoptosis in various tumor cells in vitro. Here, we investigated the effect of the natural alkaloid berberine on As2O3-mediated inhibition of cancer cell migration using rat and human glioma cell lines.MethodsThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of rat C6 and human U-87 glioma cells after treatment with As2O3 or berberine, and after co-treatment with As2O3 and berberine. The wound scratch and Boyden chamber assays were applied to determine the effect of As2O3 and berberine on the migration capacity and invasiveness of glioma cancer cells. Zymography and Western blot analyses provided information on the effect of As2O3 and berberine on the intracellular translocation and activation of protein kinase C (PKC), and some PKC-related downstream factors. Most assays were performed three times, independently, and data were analyzed using ANOVA.ResultsThe cell viability studies demonstrated that berberine enhances As2O3-mediated inhibition of glioma cell growth after 24 h incubation. Untreated control cells formed a confluent layer, the formation of which was inhibited upon incubation with 5 μM As2O3. The latter effect was even more pronounced in the presence of 10 μM berberine. The As2O3-mediated reduction in motility and invasion of glioma cells was enhanced upon co-treatment with berberine. Furthermore, it has been reported that PKC isoforms influence the morphology of the actin cytoskeleton, as well as the activation of metalloproteases MT1-MMP and MMP-2, reported to be involved in cancer cell migration. Treatment of glioma cells with As2O3 and berberine significantly decreased the activation of PKC α and ε and led to actin cytoskeleton rearrangements. The levels of two downstream transcription factors, myc and jun, and MT1-MMP and MMP-2 were also significantly reduced.ConclusionUpon co-treatment of glioma cells with As2O3 and berberine, cancer cell metastasis can be significantly inhibited, most likely by blocking the PKC-mediated signaling pathway involved in cancer cell migration. This study is potentially interesting for the development of novel chemotherapeutic approaches in the treatment of malignant gliomas and cancer development in general.

Is the endogenous peroxyl-radical scavenging capacity of plasma protective in systemic inflammatory disorders in humans?

Systemic inflammatory response syndrome (SIRS) in humans is associated with heightened intravascular oxidative stress. The clinical significance of plasma endogenous antioxidative capability in SIRS remains undetermined. Time-sequence changes of plasma total radical-trapping antioxidant parameter (TRAP) and its components were measured in 135 patients with various clinical conditions leading to SIRS. The results were correlated with clinical parameters. Plasma TRAP significantly depressed upon diagnosis of SIRS (SIRS vs. healthy subjects (n = 50), 605.7 +/- 20.4 vs. 803.4 +/- 30.8 microM Trolox equivalent, p <.001). In survivors (n = 86), TRAP declined further during the course of SIRS, followed by a mild recovery at the end of follow-up. General linear mixed model analysis revealed that uric acid, vitamin C, vitamin E and unidentified antioxidants contributed to most of the changes in TRAP (each factor p <.001). In nonsurvivors (n = 49), TRAP increased steadily until death, and the increase was predominantly the result of the increased contribution of bilirubin (p <.01). Higher TRAP levels were not correlated with diminished blood oxidants formation (r = -0.13, p.05), lower intensity of lipid peroxidation (r = 0.261, p <.05) or lesser disease severity of SIRS. The results do not support the hypothesis that the endogenous peroxyl radical scavenging ability of plasma plays a protective role in the course of SIRS.

Loss of heterozygosity and microsatellite instability in hepatocellular carcinoma in Taiwan

SummaryElucidation of the basic genetic changes of human hepatocellular carcinoma is important for the understanding and treatment of this cancer. We used microsatellite polymorphism markers to study 30 cases of hepatocellular carcinoma (34 tumours) on all human chromosomes. DNA from 34 pairs of hepatocellular carcinomas and corresponding non-tumour parts was prepared. Loss of heterozygosity (LOH) and microsatellite instability on 23 chromosomes were investigated by 231 sets of microsatellite markers. More than 20% LOH was shown for loci on 16q (47.1%), 13q (32.4%), 17p (32.4%), 5q (26.5%), 11p (23.5%) and 9p (20.6%). The commonly affected regions were mapped to 16q12.1, 16q12.2, 16q24, 13q12.1–32, 17p13, 5q32, 5q34, 5q3, 11p15, 11q23–24 and 9p21. Hepatitis B virus carriers had a significantly higher frequency of LOH on chromosomes 5q, 11p and 16q. Furthermore, larger tumour size tended to have higher frequency of LOH at D16S409 locus (16q12.1). Microsatellte instability was only found in 12 of 231 markers and the frequency is very low. These data suggest that the chromosomes 16q, 13q, 17p, 5q, 11p and 9p might participate in hepatocarcinogenesis. However, microsatellite instability might play little role in the development of this cancer in Taiwan.

Protective effects of Dunaliella salina : A carotenoids-rich alga, against carbon tetrachloride-induced hepatotoxicity in mice

The protective effects of Dunaliella salina (D. salina) on liver damage were evaluated by carbon tetrachloride (CCl(4))-induced hepatotoxicity in mice. Male ICR mice were orally treated with D. salina or silymairn daily with administration of CCl(4) twice a week for 8 weeks. CCl(4) induced liver damage and significantly (p<0.05) increased the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in serum and decreased the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), and GSH content in liver whereas increased hepatic malondialdehyde (MDA) content as compared with control group. Treatment with D. salina or silymarin could significantly (p<0.05) decrease the ALT, AST, and ALP levels in serum and increase the activities of SOD, catalase, GSH-Px, glutathione reductase, and GSH content and decrease the MDA content in liver when compared with CCl(4)-treated group. Liver histopathology also showed that D. salina reduced the incidence of liver lesions induced by CCl(4). The results suggest that D. salina exhibits potent hepatoprotective effects on CCl(4)-induced liver damages in mice, and that the hepatoprotective effects of D. salina may be due to both the increase of antioxidant enzymes activities and inhibition of lipid peroxidation.

Hepatoprotective effect of electrolyzed reduced water against carbon tetrachloride-induced liver damage in mice.

The study investigated the protective effect of electrolyzed reduced water (ERW) against carbon tetrachloride (CCl(4))-induced liver damage. Male ICR mice were randomly divided into control, CCl(4), CCl(4)+silymarin, and CCl(4)+ERW groups. CCl(4)-induced liver lesions include leukocytes infiltration, hepatocyte necrosis, ballooning degeneration, mitosis, calcification, fibrosis and an increase of serum alanine aminotransferase (ALT), and aminotransferase (AST) activity. In addition, CCl(4) also significantly decreased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). By contrast, ERW or silymarin supplement significantly ameliorated the CCl(4)-induced liver lesions, lowered the serum levels of hepatic enzyme markers (ALT and AST) and increased the activities of SOD, catalase, and GSH-Px in liver. Therefore, the results of this study show that ERW can be proposed to protect the liver against CCl(4)-induced oxidative damage in mice, and the hepatoprotective effect might be correlated with its antioxidant and free radical scavenging effect.