Tsan-Zon Liu

Homocysteine thiolactone induces apoptotic DNA damage mediated by increased intracellular hydrogen peroxide and caspase 3 activation in HL-60 cells

The cytotoxicity of homocysteine derivatives on chromosomal damage in somatic cells is not well established. The present study used reactive homocysteine derivative of homocysteine thiolactone (Hcy) to investigate its causal effect on apoptotic DNA injury in human promyeloid HL-60 cells. Our results demonstrated that Hcy induced cell death and features of apoptosis including increased phosphotidylserine exposure on the membrane surface, increased apoptotic cells with hypoploid DNA contents, and internucleosomal DNA fragmentation, all of which occurred in a time- and concentration-dependent manner. Hcy treatment also significantly increased intracellular reactive oxygen species H2O2, which coincided with the elimination of caspase 3 proenzyme levels and increased caspase 3 activity at the time of the appearance of apoptotic DNA fragmentation. Preincubation of Hcy-treated HL-60 cells with catalase completely scavenged intracellular H2O2, thus inhibiting caspase 3 activity and protecting cells from apoptotic DNA damage. In contrast, superoxide dismutase failed to inhibit Hcy-induced DNA damage. Taken together, these results demonstrate that Hcy exerted its genotoxic effects on HL-60 cells through an apoptotic pathway, which is mediated by the activation of caspase 3 activity induced by an increase in intracellular hydrogen peroxide.

Evidence for accelerated generation of hydroxyl radicals in experimental obstructive jaundice of rats.

We present evidence herein of the accelerated generation of hydroxyl radical (.OH) in the plasma and the liver tissue of common bile duct ligated (CBDL) rats, a model for experimental obstructive jaundice. .OH production in the plasma was monitored in vivo by the identification of dihydroxybenzoates in plasma [2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (2,5-DHB)] using high performance liquid chromatography (HPLC). The average concentrations of 2,3-DHB and 2,5-DHB produced in the plasma of the controls were 33+/-3 microM and 232+/-34 microM (n = 15), respectively, whereas their respective concentrations increased to 149+/-28 microM and 604+/-88 microM in the CBDL rats (n = 19). Furthermore, we also observed a time-dependent decreasing trend of 2,3-DHB and 2,5-DHB production after surgical removal of the ligation of the experimental animals. In addition, the generation of .OH in the liver tissue was studied by using dimethyl sulfoxide (DMSO) as a molecular probe and measuring the amount of methanesulfinic acid (MSA), the product of the trapping reaction. The net production of MSA in the liver tissue of the control rats was 1.22+/-0.05 O.D. unit/g protein (n = 5), whereas its respective concentration of MSA in the liver tissue of CBDL rats increased to 2.05+/-0.15 O.D. unit/g protein (n = 5). In addition, we showed that CBDL rats receiving a pretreatment of mannitol, an .OH scavenger, resulted in the decreased production of MSA. Electron micrographic study indicated that the most prominent change observed in CBDL rats was the alteration of mitochondria, which were swollen with distorted cristae. Meanwhile, the bile canaliculi were moderately more dilated than that of the controls, and an increased neutrophil peripheral blood count was found in CBDL rats when compared to the controls. Taken together, our data suggest that accelerated generation of .OH in the CBDL rats is obvious and may play a key role in the pathogenesis of liver damage associated with obstructive jaundice.

Correlation of membrane lipid peroxidation with oxidation of hemoglobin variants: possibly related to the rates of hemin release.

Experiments were performed to delineate the biochemical mechanism of hemoglobin (Hb)-catalyzed lipid peroxidation in human red blood cells (RBCs). Using a modified Langmuir trough lipid monolayer technique, we found that oxidized Hb induced an increase in lipid monolayer surface pressure, suggesting that oxidized Hb readily releases its heme moiety into the lipid monolayer. To confirm our interpretation that oxidized Hb readily releases its heme moiety, we monitored the fluorescence of Hb tryptophan upon oxidation of Hb. We found an increase in Hb fluorescence in the aqueous phase of our monolayer system after the addition of H2O2. The increase in fluorescence should reflect the departure of heme from globin due to a decrease in fluorescent quenching effect by the heme moiety. The rate of increase in lipid monolayer surface pressure upon Hb oxidation differed from Hb to Hb with an order of Hb E > F > S > A. The ability of various Hbs to affect lipid peroxidation in the RBC membrane, as monitored by the parinaric acid oxidation technique, followed this same order. In addition, hemin was shown to be a more potent catalyst of lipid peroxidation in RBC membrane than nonheme irons.

Differentiation status modulates transcription factor NF-κB activity in unstimulated human hepatocellular carcinoma cell lines

We report herein a novel finding that under an unstimulated condition, a group of four human hepatocellular carcinoma (HCC) cell lines with varying degrees of differentiation, can spontaneously activate NF-KB. The propensity of activation coincided inversely with the differentiation status, with order being SK-Hep-1 > J5 > Hep3B > HepG2. Further studies indicate that this pattern of activation correlates excellently with the descending order of intracellular GSH/GSSG ratios as well as with the ascending order in the ability of these cells to generate hydrogen peroxide. Taken together, our data suggest that differentiation status may play a pivotal role in modulating intracellular thiol redox status and the extent of catalase expression, which may be crucial in the control of NF-kappaB activity in these HCC cells.

(−)-Anonaine induces apoptosis through Bax- and caspase-dependent pathways in human cervical cancer (HeLa) cells.

(-)-Anonaine has been shown to have some anticancer activities, but the mechanisms of (-)-anonaine inducing cell death of human cancer cells is not fully understood. We investigated the mechanisms of apoptosis induced by (-)-anonaine in human HeLa cancer cells. Treatment with (-)-anonaine induces dose-dependent DNA damage that is correlated with increased intracellular nitric oxide, reactive oxygen species, glutathione depletion, disruptive mitochondrial transmembrane potential, activation of caspase 3, 7, 8, and 9, and poly ADP ribose polymerase cleavage. Our data indicate that (-)-anonaine up-regulated the expression of Bax and p53 proteins in HeLa cancer cells. The apoptosis and expression of Bax induced by (-)-anonaine could be inhibited when the HeLa cells were pretreated with Boc-Asp(OMe)-fmk, which is a broad caspases inhibitor. There was no obvious DNA damage in the (-)-anonaine-treated Madin-Darby canine kidney and Vero cell lines. Both Madin-Darby canine kidney and Vero cell lines are kidney epithelial cellular morphology. These results suggest that (-)-anonaine might be considered a potent compound for chemotherapy against cervical cancer or a health food supplement for cancer chemoprevention.

The isoprostanes: unique bioactive products of lipid peroxidation. An overview.

The development of a specific, reliable and noninvasive method for measuring oxidative stress in humans is essential for establishing the role of free radicals in human diseases. Currently, accurate techniques to assess oxidant injury in vivo are extremely limited although a number of approaches are being investigated. Of these, the measurement of specific products of nonenzymatic lipid peroxidation, the F2-isoprostanes (F2-IsoPs), appears to be a more accurate marker of oxidative stress in vivo in humans than other available methods. The purpose of this brief review is to acquaint the reader with the IsoPs from a biochemical perspective and to provide information regarding the utility of quantifying these compounds as indicators of oxidant stress.

Humic acid induces the generation of nitric oxide in human umbilical vein endothelial cells: stimulation of nitric oxide synthase during cell injury.

Humic acid (HA) has been implicated as an etiological factor in the peripheral vasculopathy of blackfoot disease (BFD). In this study, we examined the effects of HA upon the generation of nitric oxide (NO) during the process of lethal cell injury in cultured human umbilical vein endothelial cells (HUVECs). NO production was measured by the formation of nitrite (NO(2)(-)), the stable end-metabolite of NO. Cell death was assessed by measuring the release of intracellular lactate dehydrogenase (LDH). Treatment HUVECs with HA at a concentration of 50, 100, and 200 microg/ml concentration-dependently increased nitrite levels, reaching a peak at 12 h subsequent to HA treatment, with a maximal response of approximately 400 pmole nitrite (from 1 x 10(4) cells). HA-induced nitrite formation was blocked completely by N(G)-nitro-L-arginine methyl ester (L-NAME) and also by N(G)-methyl-L-arginine (L-NMA), both being specific inhibitors of NO synthase. The LDH released from endothelial cells was evoked at from 24 h after the addition of HA (50, 100, 200 microg/ml) in a concentration- and time-dependent manner. The HA-induced LDH release was also reduced by the presence of both L-NAME and L-NMA. The addition of Ca(2+) chelator (BAPTA) inhibited both nitrite formation and LDH release by HA. Moreover, the antioxidants (superoxide dismutase, vitamin C, vitamin E) and protein kinase inhibitor (H7) effectively suppressed HA-induced nitrite formation. These results suggest that HA treatment of endothelial cells stimulates NO production, which can elicit cell injury via the stimulation of Ca(2+)-dependent NO synthase activity by increasing cytosolic Ca(2+) levels. Because the destruction of endothelial cells has been implicated in triggering the onset of BFD, the induction of excessive levels of NO and consequent endothelial-cell injury may be important to the etiology of HA-induced vascular disorders associated with BFD for humans.

Antimutagenicity of ethanol extracts of bee glue against environmental mutagens.

The antimutagenicity of ethanol extracts of bee glue (propolis) (EEBG) was evaluated, using Salmonella typhimurium strain TA98 as a test model, against two direct mutagens, 4-nitro-O-phenylenediamine (4-NO) and 1-nitropyrene (1-NP), and two indirect mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and benzo[a]pyrene (B[a]P) with S9 mix. EEBG was shown to suppress the mutagenicity of these compounds in a dose-dependent fashion. To delineate the mechanism of action of the antimutagenic effects of EEBG on the two indirect mutagens IQ and B[a]P, two possible points of blocking were considered: (1) cytochrome P-450 activity (route 1) and (2) interaction with microsome-generated proximate mutagens to generate an inactive complex (route 2). Our results clearly demonstrated, at a very low dose, remarkable suppression of the mutagenicity of both compounds by inhibiting either route 1 or route 2 pathway. Further studies indicated that EEBG was capable of inhibiting both the activities of hepatic cytochrome P-450 IA1-linked 7-ethoxyresorufin-O-deethylase (EROD) and IA 2-linked 7-ethoxycoumarin-O-deethylase (ECD) in a similar dose-dependent manner. Taken together, we demonstrated that EEBG was a good inhibitor for mutagenicity of direct mutagens, 1-NP and 4-NO, as well as for the indirect mutagens IQ and B[a]P in the presence of S9 mix via inactivation of microsomal enzyme activities (e.g. EROD and ECD) or antagonizing metabolic generation of the proximate mutagens of IQ and B[a]P.

The Isoprostanes: Unique Bioactive Products of Lipid Peroxidation

The development of a specific, reliable and noninvasive method for measuring oxidative stress in humans is essential for establishing the role of free radicals in human diseases. Currently, accurate techniques to assess oxidant injury in vivo are extremely limited although a number of approaches are being investigated. Of these, the measurement of specific products of nonenzymatic lipid peroxidation, the F2-isoprostanes (F2-IsoPs), appears to be a more accurate marker of oxidative stress in vivo in humans than other available methods. The purpose of this brief review is to acquaint the reader with the IsoPs from a biochemical perspective and to provide information regarding the utility of quantifying these compounds as indicators of oxidant stress.

Enhanced susceptibility of erythrocytes deficient in glucose-6-phosphate dehydrogenase to alloxan/glutathione-induced decrease in red cell deformability.

It has been hypothesized that enhanced oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD) deficient red cells(RBCs) is the underlying mechanism for drug- or chemical-induced hemolytic crises in G6PD-deficiency. To further test this hypothesis, we used an alloxan-glutathione system to mimic oxidative stress and see how oxidative damage might affect RBC deformability. RBC deformability, a major determinant of RBC survival in vivo, was monitored by a laser viscodiffractometer. Under our experimental conditions, GSH alone had very little effect on the deformability of either normal or G6PD-deficient RBCs. In contrast, alloxan alone induced a small but significant decrease in the deformability of either normal or G6PD-deficient RBCs. Interestingly, alloxan and GSH together induced a further decrease in the deformability of either normal or G6PD-deficient RBCs. The decrease in deformability in G6PD-deficient RBCs was much more profound than in normal RBCs. In addition, an alloxan-vitamin C system produced a similar deleterious effect on RBC deformability as that produced by the alloxan-GSH system. Appreciable amount of hydroxyl radicals was generated by both alloxan-GSH and alloxan-vitamin C systems as evidenced by the production of hydroxylated products of salicylate which was used as a radical trap. Moreover, salicylate could ameliorate the deleterious effect of the alloxan system on the deformability of RBCs. Taken together, our results demonstrated that G6PD-deficient RBCs were particularly susceptible to oxidant-induced damage leading to a dramatic decrease in their deformability and thus provided strong support for the hypothesis that enhanced oxidant sensitivity of G6PD-deficient RBCs is the underlying mechanism for accelerated destruction of these RBCs in vivo.