Mentha pulegium crude extracts induce thiol oxidation and potentiate hemolysis when associated to t-butyl hydroperoxide in human's erythrocytes

ABSTRACT Mentha pulegium (Lamiaceae) tea has been used as a traditional medicine; however, the modulatory effect of M. pulegium extracts on damage to human erythrocytes associated to t-butyl hydroperoxide (t-BHP) exposure remains to be investigated. Accordingly, we perform this study in order to test the hypothesis that aqueous and ethanolic extracts of M. pulegium could modulate the hemolysis associated to t-BHP exposure, non-protein thiol (NPSH) oxidation and lipid peroxidation (measured as thiobarbituric acid reactive substances - TBARS) in human erythrocytes. Samples were co-incubated with t-BHP (4 mmol/L) and/or aqueous or ethanolic extracts (10-1000 mg/mL) during 120 min to further analysis. We found that both extracts, when associated to t-BHP, potentiate NPSH oxidation and hemolysis. Moreover, both extracts significantly prevents against t-BHP-induced TBARS production. A significant correlation among hemolysis and NPSH levels was found. Taking together, our data points that the association of M. pulegium extracts with t-BHP culminates in toxic effect to exposed erythrocytes, besides its protective effect against t-BHP-induced TBARS production. So, we infer that the use of this extract may exert negative effect during painful crisis in sickle cell anemia. However, more studies are still necessary to better investigate/understand the mechanism(s) involved in the toxic effect resultant from this association.

The effects of hydromorphone on astrocytic responses in cerebral ischemia

BACKGROUND: Ischemic insult during operation could cause ischemic-reperfusion injuries in brain and memory impairments. Total intravenous anesthesia (TIVA) is preferred in brain surgery to promote the use of motor evoked potential monitoring and the use of opioids is common in TIVA. However there were few studies about ischemic protective effect of opioids to astrocytes. METHODS: We used astrocytes, which were derived from human brain. We divided groups by conditioning period; i) pre-culture, ii) post-culture, or iii) pre + post-culture. All groups were treated 100 nM hydromorphone. We measured reactive oxygen species (ROS) by flow cytometry with 2',7'-dichloroflurorescin diacetate. Then ROS in astrocytes which treated by opioid receptor antagonist were measured after treating 100 nM hydromorphone. RESULTS: ROS was reduced in hydromorphone treated group, as compared to the control group (only tert-butyl hydroperoxide [TBH] treated). There was no difference in pre-conditioned group and post-conditioned group. However, ROS was much more reduced in pre + post-conditioned group compared to pre-conditioned only or post-conditioned only group. Furthermore each selective micro-, delta- and kappa-opioid receptor antagonists partially negated the effect of hydromorphone. CONCLUSIONS: This study provides evidence that hydromorphone has both preconditioning and postconditioning effects on TBH-induced oxidative stress. Furthermore we proved each micro-, delta- and kappa-opioid receptor relates to protective mechanism of hydromorphone to astrocytes.

Black rice extract protected HepG2 cells from oxidative stress-induced cell death via ERK1/2 and Akt activation

BACKGROUND/OBJECTIVES: The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS: Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS: TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS: These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress.

Schisandra Chinensis Baillon regulates the gene expression of phase II antioxidant/detoxifying enzymes in hepatic damage induced rats

BACKGROUND/OBJECTIVES: This study investigated the antioxidant activities and hepatoprotective effects of Schisandra chinensis Baillon extract (SCE) against tert-butyl hydroperoxide (t-BHP)-induced oxidative hepatic damage in rats. MATERIALS/METHODS: Sprague-Dawley (SD) rats were pretreated with SCE (300, 600, and 1,200 mg/kg BW) or saline once daily for 14 consecutive days. On day 14, each animal, except those belonging to the normal control group, were injected with t-BHP (0.8 mmol/kg BW/i.p.), and all of the rats were sacrificed 16 h after t-BHP injection. RESULTS: Although no significant differences in AST and ALT levels were observed among the TC and SCE groups, the high-dose SCE group showed a decreasing tendency compared to the TC group. However, erythrocyte SOD activity showed a significant increase in the low-dose SCE group compared with the TC group. On the other hand, no significant differences in hepatic total glutathione (GSH) level, glutathione reductase (GR), and glutathione peroxidase (GSH-Px) activities were observed among the TC and SCE groups. Hepatic histopathological evaluation revealed that pretreatment with SCE resulted in reduced t-BHP-induced incidence of lesions, such as neutrophil infiltration, swelling of liver cells, and necrosis. In particular, treatment with a high dose of SCE resulted in induction of phase II antioxidant/detoxifying enzyme expression, such as glutathione S-transferase (GST) and glutamate-cysteine ligase catalytic subunit (GCLC). CONCLUSIONS: Based on these results, we conclude that SCE exerts protective effects against t-BHP induced oxidative hepatic damage through the reduction of neutrophil infiltration, swelling of liver cells, and necrosis. In addition, SCE regulates the gene expression of phase II antioxidant/detoxifying enzymes independent of hepatic antioxidant enzyme activity.

Effect of Dipyridamole on the Reactive Oxygen Species and Oxidative Stress in Trabecular Meshwork Cells

PURPOSE: To investigate the effects of dipyridamole (DPD) on the production of reactive oxygen species (ROS) and oxidative stress in cultured human trabecular meshwork cells (HTMC). METHODS: Antioxidant activity of DPD was determined by DPPH assay. Primarily cultured HTMC were exposed to 0, 20, and 50 microm DPD using serum-deprived media. The effect of DPD on the production of ROS was assessed with the DCHFDA assay. The effect of DPD on the t-butyl hydroperoxide (tBHP)-induced oxidative stress was assessed with resazurin assay. RESULTS: DPD showed significant antioxidant activity. DPD significantly decreased the production of ROS (p < 0.05) and improved cellular activity significantly after treatment with t-BHP (p < 0.05). DPD did not affect the generation of nitric oxides. CONCLUSIONS: DPD suppressed the formation of ROS and possessed cytoprotective activity against the oxidative stress in HTMC.

Contradictory Effects of Superoxide and Hydrogen Peroxide on KCa3.1 in Human Endothelial Cells

Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. KCa3.1 plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on KCa3.1 expression and the K+ current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated KCa3.1 expression, while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysopho-sphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, KCa3.1 current, which was activated by clamping cells with 1 microM Ca2+ and applying the KCa3.1 activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP, and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases KCa3.1 expression by upregulating pERK and downregulating REST, and augments the K+ current. On the other hand, superoxide reduces KCa3.1 expression by downregulating pERK and upregulating REST, and inhibits the K+ current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating KCa3.1 and endothelial function.

Regulatory effect of miR-181a on expression of c-fos in cochlear hair cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the regulatory effect of miR-181a with abnormal expression on the expression of c-fos in cochlear hair cells undergoing oxidative damage.</p><p><b>METHODS</b>House Ear Institute-Organ of Corti1 (HEI-CO1) cells were assigned to 50, 100, and 200 µmol/L tert-butyl hydroperoxide (t-BHP) exposure groups and control group. The HEI-CO1 cells in the exposure groups were exposed to 50, 100, or 200 µmol/L t-BHP for 12 h. Then, total RNA and total protein were extracted from the HEI-CO1 cells, and the expression of miR-181a/-181d was measured by qPCR. The miR-181a with abnormal expression was selected as the subject of study. The putative miR-181a target sequence in the 3' untranslated region (3'-UTR) of c-fos was predicted by searching on a bioinformatics website. The HEI-CO1 cells were transfected with miR-181a mimics by lipofection, and the transfection efficiency was measured by qPCR. The mRNA and protein expression of c-fos was measured by qPCR and Western blot. The pGL3-c-fos-3'UTR-WT plasmid was constructed, and the luciferase activity of the plasmid in the case of high miR-181a expression was measured using the Dual-Luciferase Reporter Assay System.</p><p><b>RESULTS</b>Compared with those in the control group, the expression of miR-181a in 100 and 200 µmol/L t-BHP exposure groups was significantly decreased, with expression ratios of 0.744 and 0.766 (P < 0.01), while the expression of miR-181d in 50 µmol/L t-BHP exposure group was significantly increased, with an expression ratio of 1.29 (P < 0.01). There was no significant difference in miR-181a expression between the 100 and 200 µmol/L t-BHP exposure groups (P > 0.05). The predication results revealed that c-fos was regulated by miR-181a in humans and mice, with complete complementarity to the seed region of miR-181a, and there was high degree of target sequence conservation across species. The expression of miR-181a in the HEI-OC1 cells transfected with miR-181a mimics was elevated 892.979 times at 24 hours after transfection. As compared with those of controls, the mRNA and protein expression levels of c-fos in the transfected HEI-OC1 cells were significantly increased (P < 0.05 and P < 0.01). The luciferase activity of pGL3-c-fos-3'UTR-WT plasmid was not suppressed but increased in the case of high miR-181a expression.</p><p><b>CONCLUSION</b>miR-181a has no direct inhibitory effect on the mRNA and protein expression of c-fos, which may not be the target gene of miR-181a. Bioinformatic prediction might produce false-positive results.</p>

Increased oxidative damages of erythrocytes caused by declined blood oxygen saturation / 生物医学工程学杂志

This paper was to explore the effect of blood oxygen saturation (SO2) on oxidative damages of erythrocytes under the condition of oxidative stress. Keeping SO2 of cultured erythrocytes in vitro at the states of 0.3, 0.5, 0.7, 0.9 and 0.98, respectively, we induced oxidative stress by tert-buthylhydroperoxide (BHP, 0.15 mmol/L of final concentration). After incubation, antioxidant capacity was assessed by measuring content of reduced glutathin hormone (GSH) in erythrocytes. Methemoglobin (MetHb) content, lipid peroxidation (thiobarbituric acid-reactive substances, TBARS) and denatured globin-chains on the plasma membrane were measured to assess the extent of oxidative damages. The results showed that in the presence of BHP, GSH contents increased from 0.3 to 0.98 groups; MetHb, TBARS and globin-chains levels all dropped with the rise of SO2. In conclusion, antioxidant capacity and oxidative damages of erythrocytes are closely related to SO2, declined SO2 could promote oxidative damages of erythrocytes.

The effects of oxidative damage induced by organic oxidant t-BHP on cochlear hair cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To establish the oxidative damage model of cochlea hair cells using organic oxidant t-BHP in vitro.</p><p><b>METHODS</b>HEI-OC1 cells were exposed to t-BHP at 8 doses (30~4000 µmol/L) for 12 h. Trypan blue test was used to detected the cellular viability and MTT assay was utilized to measured the cellular proliferation. The intracellular ROS levels were determined by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA).</p><p><b>RESULTS</b>The survival rates of HEI-OC1 cells started decrease significantly at the dose of 100 µmol/L t-BHP, the peak of decreased survival rates appeared at the doses of 200~800 µmol/L. The results of MTT assay demonstrated that 30 µmol/L t-BHP could promote cellular proliferation ability, when t-BHP concentrations were higher than 200 µmol/L, the cellular proliferation ability was inhibited. The results of DCFH-DA assay showed that there was no fluorescence in control group, the strong fluorescence was observed in positive control group, the weak fluorescence was observed in 30~50 µmol/L t-BHP groups, the bright fluorescence was observed in 100 µmol/L t-BHP group, still the stronger fluorescence was observed in 200~1000 µmol/L groups, but the cellular number decreased with the doses because of the lower cellular viability.</p><p><b>CONCLUSION</b>The exposure to 100 µmol/L t-BHP for 12 h could simulate the oxidative damage induced by noise in cochlear hair cells.</p>

Desalinated underground seawater of Jeju Island (Korea) improves lipid metabolism in mice fed diets containing high fat and increases antioxidant potential in t-BHP treated HepG2 cells

This study was performed to investigate the effect of desalinated underground seawater (named as 'magma seawater', MSW) of Jeju Island in Korea on lipid metabolism and antioxidant activity. MSW was collected from underground of Han-Dong in Jeju Island, and freely given to high fat diet (HFD)-fed C57BL/6 mice for 10 weeks. Although there were no significant differences in the body weight changes and plasma lipid levels, hepatic triglyceride levels were significantly lower in the MSW group than in the normal tap water (TW)-drunken control group. Furthermore, the activity of fatty acid synthase (FAS) was significantly decreased and carnitine palmitoyltransferase (CPT) activity was increased in MSW group compared to TW group. Similarly, real-time PCR analysis revealed that mRNA expressions of lipogenic genes were lowered in MSW groups compared to the control group. In a morphometric observation on the liver tissue, accumulation of fats was remarkably reduced in MSW group. Meanwhile, in vitro assay, free radical scavenging activity measured by using diphenylpicrylhydrazyl (DPPH) was increased in MSW group. The 2'-7'-dichlorofluorescein diacetate (DCF-DA) staining followed with fluorescent microscopy showed a low intensity of fluorescence in MSW-treated HepG2 cells, compared to TW-treated HepG2 cells, which indicated that the production of reactive oxygen species by tert-butyl hydroperoxide (t-BHP) in HepG2 cells was decreased by MSW treatment. The antioxidant effect of MSW on t-BHP-induced oxidative stress in HepG2 cells was supported by the increased activities of intracellular antioxidant enzymes such as catalase and glutathione reductase. From these results, we speculate that MSW has an inhibitory effect on lipogenesis in liver and might play a protective role against cell damage by t-BHP-induced oxidative stress.

Kinetics of oxidation of adenosine by tert-butoxyl radicals: Protection and repair by chlorogenic acid.

The rates of oxidation of adenosine and chlorogenic acid by tert-butoxyl radicals (t-BuO-) were studied by measuring the absorbance of adenosine at 260 nm and chlorogenic acid at 328 nm spectrophotometrically. t-BuO- radicals were generated by the photolysis of tert-butyl hydroperoxide (t-BuOOH) in presence of tert-butyl alcohol to scavenge OH. radicals. The rates and the quantum yields() of oxidation of chlorogenic acid by t-BuO-radicals were determined in the absence and presence of varying concentrations of adenosine. An increase in the concentration of adenosine was found to decrease the rate of oxidation of chlorogenic acid, suggesting that adenosine and chlorogenic acid competed for t-BuO. radicals. From competition kinetics, the rate constant of chlorogenic acid reaction with t-BuO- was calculated to be 3.20 109 dm3 mol-1 s-1. The quantum yields (expt) were calculated from the experimentally determined rates of oxidation of chlorogenic acid under different experimental conditions. Assuming that chlorogenic acid acts as a scavenger of t-BuO- radicals only, the quantum yields (cal) were theoretically calculated. expt and cal values suggested that chlorogenic acid not only protected adenosine from t-BuO- radicals, but also repaired adenosine radicals, formed by the reaction of adenosine with t-BuO- radicals.

Antioxidant potential of the root of Vetiveria zizanioides (L.) Nash.

Vetiveria zizanioides, an aromatic plant commonly known as vetiver has been used for various ailments. The essential oil of vetiver root has been shown to possess antioxidant activity. However, antioxidant potential of spent root extract has not been reported. Hence, in the present study, ferric reducing, free radical scavenging and antioxidant activity of two genotypes namely KS1 and gulabi of V. zizanioides L. Nash root were investigated using in vitro assays - the ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), total phenolic content (TPC), total antioxidant capacity (TAC) and reducing power (RP). KS1 genotype showed higher FRAP values, DPPH inhibition, TPC and RP potential compared to gulabi and the antioxidant activity increased with the concentration of the extract (10-1000 microg/mL). A significant protective effect of cv KS1 (100 microg/mL) extract was also observed in reduced glutathione (GSH) and malondialdehyde (MDA) concentrations of erythrocytes subjected to oxidative stress by tert-butyl hydroperoxide (t-BHP) and hydrogen peroxide (H202). The cv KS1 showed better antioxidant activity, compared to cv gulabi indicating the possibility of exploring the presence of different phytoconstituents in the two varieties.

Oxidative stress induces apoptosis via NF-kappaB-iNOS-nitric oxide pathway in pancreatic beta-cells / 中国应用生理学杂志

<p><b>AIM</b>To explore the possible mechanism of tert-butyl hydroperoxide (t-BHP)-induced apoptosis in murine MIN6 pancreatic beta-cells.</p><p><b>METHODS</b>MIN6 cells were cultured in vitro. Cell damage was evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of cell apoptosis was determined by flow cytometric assay after Annexin- V-PI staining. Nitric oxide levels were measured by Griess assay. Inducible nitric oxide synthase(iNOS) protein and NF-kappaBp65 fragment were detected by Western blot.</p><p><b>RESULTS</b>Exposure of 25 micromol/L t-BHP to MIN6 cells for 60 min, cell viability was reduced and the percentage of apoptosis was increased significantly. The levels of cytoplasmic iNOS protein and nitrite were elevated. Meanwhile, treatment with t-BHP resulted in nucleus NF-kappaBp65 fragment peaking at 20 min. Both L-NAME and N-Acetyl-l-cysteine (NAC) attenuated the elevated levels of nitrite and percentage of apoptosis due to t-BHP alone.</p><p><b>CONCLUSION</b>NF-kappa-iNOS-nitric oxide signalling pathway can mediated t-BHP induced apoptosis in MIN6 cells .</p>

High susceptibility of activated lymphocytes to oxidative stress-induced cell death

The present study provides evidence that activated spleen lymphocytes from Walker 256 tumor bearing rats are more susceptible than controls to tert-butyl hydroperoxide (t-BOOH)-induced necrotic cell death in vitro. The iron chelator and antioxidant deferoxamine, the intracellular Ca2+ chelator BAPTA, the L-type Ca2+ channel antagonist nifedipine or the mitochondrial permeability transition inhibitor cyclosporin A, but not the calcineurin inhibitor FK-506, render control and activated lymphocytes equally resistant to the toxic effects of t-BOOH. Incubation of activated lymphocytes in the presence of t-BOOH resulted in a cyclosporin A-sensitive decrease in mitochondrial membrane potential. These results indicate that the higher cytosolic Ca2+ level in activated lymphocytes increases their susceptibility to oxidative stress-induced cell death in a mechanism involving the participation of mitochondrial permeability transition.

O presente estudo demonstra que linfócitos ativados de baço de ratos portadores do tumor de Walker 256 são mais susceptíveis à morte celular necrótica induzida por tert-butil hidroperóxido (t-BOOH) in vitro quando comparados aos controles. O quelante de ferro e antioxidante deferoxamina, o quelante intracelular de Ca2+ BAPTA, o antagonista de canal de Ca2+ nifedipina ou o inibidor da transição de permeabilidade mitocondrial ciclosporina-A, mas não o inibidor de calcineurina FK-506, inibiram de maneira similar a morte celular induzida por t-BOOH em linfócitos ativados e controles. Os linfócitos ativados apresentaram redução do potencial de membrana mitocondrial induzida por t-BOOH num mecanismo sensível a ciclosporina-A. Nossos resultados indicam que o aumento da concentração de Ca2+ citosólico em linfócitos ativados aumenta a susceptibilidade dos mesmos à morte celular induzida por estresse oxidativo, num mecanismo envolvendo a participação do poro de transição de permeabilidade mitocondrial.

Exendin-4 protected murine MIN6 pancreatic beta-cells from oxidative stress-induced apoptosis via down-regulation of NF-kappaB-iNOS-NO pathway / 药学学报

To explore the effect of glucagon-like peptide-1 receptor agonist--Exendin-4 (Ex-4) on murine MIN6 pancreatic beta-cells apoptosis induced by oxidative stress, the morphological changes of cell damage were evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of cell apoptosis was determined by flow cytometric assay after Annexin-V-FITC-PI staining. Nitric oxide level was measured by Griess reagent assay. Inducible nitric oxide synthase (iNOS) protein and NF-kappaBp65 fragment were detected by Western blotting. Ex-4 inhibited the increase of nitrite level and percentage of apoptosis induced by t-BHP in MIN6 cells. Furthermore, Ex-4 partly reduced the expression of iNOS protein and the ratio of NF-kappaBp65 protein in nucleus:cytosol induced by t-BHP. These results suggest that Ex4 protects MIN6 pancreatic kappa-cells from oxidative stress-induced apoptosis via down-regulation of NF-kappaB-iNOS-nitric oxide pathway.

Assessment of Hemorheological Deformability of Human Red Cells Exposed to tert-Butyl Hydroperoxide, Verapamil and Ascorbate by Ektacytometer / 대한진단검사의학회지

BACKGROUND: Normal erythrocyte is deformable and this facilitates blood flow in the capillaries. Oxidative stress reduces the deformability of erythrocytes, and influences on blood flow in microcirculation. The objective of this study was to investigate the deformability of erythrocytes exposed to oxidative stress, the protective effects of verapamil and ascorbic acid against oxidative damages in erythrocytes, and the value of the microfluidic ektacytometer, RheoScan-D (RheoMeditech, Korea) in clinical application. METHODS: Effects of oxidative stress on erythrocytes were investigated using tert-butyl hydroperoxide (tBHP). Before exposure to tBHP, the erythrocytes were pretreated with verapamil and ascorbic acid to examine their protective effect against oxidative damages. The deformability of erythrocytes was measured by the microfluidic ektacytometer, RheoScan-D. RESULTS: When treated with tBHP, the deformability of erythrocytes was decreased (P<0.01) and methemoglobin (metHb) formation and mean corpuscular volume (MCV) of erythrocytes were increased (P<0.01, P<0.05) compared to those of the untreated control cells. Compared to the tBHP treated cells, pretreatment with verapamil increased the deformability of erythrocytes (P<0.01) and decreased metHb formation (P<0.01) and MCV (P<0.05). Likewise, pretreatment with ascorbic acid increased the deformability of erythrocytes (P<0.01) and decreased metHb formation (P<0.01). CONCLUSIONS: Oxidative stress reduces the deformability of erythrocytes and the deformability could be one of markers for oxidative damage. Verapamil and ascorbic acid have protective role against tBHP induced oxidative stress. The ektacytometer, RheoScan-D used in this study is convenient for clinical measurement and could be used in various fields of clinical medicine.

Reaction of substituted pyrimidines with photochemically generated t-BuO* radicals.

Humans are exposed to various organic peroxides through chemical, pharmaceutical and cosmetic products. On photolysis, these peroxides produce alkoxyl radicals and hydroxyl radicals. The reaction of *OH radicals with DNA and its constituents have been extensively studied, but very little is known about the reactions of alkoxyl radicals with DNA and its constituents. In view of this, the oxidation of pyrimidine bases viz., thymine, uracil, cytosine, 5-bromouracil, 6-methyluracil and 1,3-dimethyluracil by t-BuO* radicals in aqueous solution at pH 7.5 has been carried out. The reaction between pyrimidine and t-BuO* is followed by measuring the absorbance of pyrimidine at the respective lambdamax. The rates of oxidation of pyrimidines are calculated from the plot of absorbance vs time. The rates of oxidation of pyrimidines have been found to increase with increase in [t-BuOOH], [pyrimidine] and light intensity. The quantum yields are calculated from the initial rates of oxidation of pyrimidine and the measured light intensity at 254 nm the wavelength at which t-BuOOH is activated to give radicals. The quantum yields are found to depend on [pyrimidine] as well as on [t-BuOOH] while they are independent of light intensity. The product analysis was carried out on HPLC with UV-visible detector. The corresponding 5,6-dihydroxypyrimidine and isobarbituric acid have been identified by comparing the retention times of the authentic samples. On the basis of experimental results and product analysis, it is suggested that t-BuOOH on photolysis gives t-BuO* radical, which initiates the reaction by adding to C (5) or C (6) position of pyrimidine base, leading to the formation of pyrimidine base radical via hydrolysis. The pyrimidine radical further reacts with t-BuO* radical to give the final product. This study predicts the probable transient pyrimidine radicals.

Hepatotoxic effects of tert-butyl hydroperoxide (t-BHP) and protection by antioxidants.

t-BHP induced oxidative stress and Ca2+ function impairment in fresh hepatocytes was studied in order to understand its role in cytotoxicity. Viability of hepatocytes by the release of lactate dehydrogenase and methyl thiazoletetrazolium reduction method alongwith malondialdehyde formation indicated oxidative stress in the hepatotoxic action of t-BHP.

Antimutagenic and antioxidant/prooxidant activity of quercetin.

The present study has been performed to evaluate the antimutagenic activity of quercetin, ascorbic acid and their combination against an oxidative mutagen. An effort was also made to correlate this activity to the in vitro antioxidant activity of these agents. Antimutagenicity testing was done in Ames Salmonella Assay system using Salmonella typhimurium TA102 against t-butylhydroperoxide as an oxidative mutagen. In vitro antioxidant scavenging activity was tested for DPPH free radical, superoxide anion, hydrogen peroxide and hydroxyl radical in their specific test systems. Quercetin (0.5-8 nmole/plate) and ascorbic acid (0.1-100 micromole/plate) showed significant effect. Quercetin (4 and 8 nmole/plate) when combined with ascorbic acid (500 nmole/plate) showed an increase in the antimutagenic activity. In vitro antioxidant activity of quercetin was better than ascorbic acid in all the test systems used. The study indicated that the antimutagenic activity of quercetin was not solely accountable by its antioxidant nature. However, in vitro free radical scavenging activity of quercetin correlated well with the antimutagenic activity.

Effect of ginsenoside Rg1 on expression of p21, cyclin E and CDK2 in the process of cell senescence / 药学学报

<p><b>AIM</b>To explore the possible role of p21, cyclin E and cyclin-dependent kinase 2 (CDK2) in the protection of ginsenoside Rg1 against tert-butylhydroperoxide (t-BHP)-induced senescence in WI-38 cells.</p><p><b>METHODS</b>The cellular ultrastructure, cytometric assay and beta-galactosidase (beta-gal) cytochemistry staining were used to evaluate cell senescence. The levels of of p21, cyclin E and CDK2 protein were detected by Western blot.</p><p><b>RESULTS</b>Pretreatment with Rg1 significantly attenuated t-BHP-induced senescence in WI-38 cells. Simultaneously, compared with cells treated with t-BHP alone, Rg1 pretreatment markedly decreased the level of p21 protein and increased the levels of CDK2 and cyclin E.</p><p><b>CONCLUSION</b>p21, cyclin E and CDK2 may be involved in the process of ginsenoside Rg1 protection against t-BHP-induced senescence in WI-38 cells.</p>