Molecular mechanism of cell death induced by the antioxidant tert-butylhydroxyanisole in human monocytic leukemia U937 cells. (33/170)

A phenolic antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) is a widely used food additive. BHA had cytotoxicity in human monocytic leukemia U937 cells. BHA at 0.75 mM caused nuclear condensation and fragmentation, structural damage in mitochondria, decrease in mitochondrial transmembrane potential, and internucleosomal DNA cleavage. It induced the activities of caspase-3 and/or -7, -6, -8 and -9, especially high when DEVD-MCA was the substrate (caspase-3 and/or -7). DEVDase activity increased in time- and dose-dependent manner and high activity was observed in lysates of cells treated for 3 h at 0.75 mM. Addition of GSH (reduced glutathione) during the treatment of cells with BHA inhibited the induction of DEVDase activity, and the intracellular GSH level decreased as the concentration of BHA was raised. Intracellular ATP levels decreased in time- and dose-dependent manner when the cells were treated with BHA in the presence or absence of glucose. Enzyme activities involved in the respiratory chain were assayed with the mitochondrial fraction prepared from U937 cells. BHA distinctly inhibited NADH-ubiquinone oxidoreductase (complex I) and cytochrome c oxidase (complex IV) at low concentrations. Succinate-ubiquinone oxidoreductase (complex II) was also inhibited, but to somewhat less extent. Without mitochondrial enzymes, BHA stimulated the ubiquinol-dependent reduction of cytochrome c (complex III), but it might have some detrimental effects on the mitochondrial enzyme reaction of complex III. The inhibition of mitochondrial oxidative phosphorylation might corroborate the mechanistic evidence for apoptosis of leukemia cells by BHA. Cell death induced by BHA is primarily ascribable to apoptosis.  (+info)

Pharmacological inhibitors of extracellular signal-regulated protein kinases attenuate the apoptotic action of cisplatin in human myeloid leukemia cells via glutathione-independent reduction in intracellular drug accumulation. (34/170)

It has been reported that inhibition of extracellular signal-regulated protein kinases (ERKs) attenuates the toxicity cisplatin (cis-platinum (II)-diammine dichloride) in some cell types. This response was here investigated using human myeloid leukemia cells. Cisplatin stimulated ERK1/2 phosphorylation and caused apoptosis in U-937 promonocytic cells, an effect which was attenuated by the MEK/ERK inhibitors PD98059 and U0126. While ERK1/2 activation was a general phenomenon, irrespective of the used cell type or antitumour drug, the MEK/ERK inhibitors only reduced cisplatin toxicity in human myeloid cells (THP-1, HL-60 and NB-4), but not in RAW 264.7 mouse macrophages and NRK-52E rat renal tubular cells; and failed to reduce the toxicity etoposide, camptothecin, melphalan and arsenic trioxide, in U-937 cells. U0126 attenuated cisplatin-DNA binding and intracellular peroxide accumulation, which are important regulators of cisplatin toxicity. Although cisplatin decreased the intracellular glutathione (GSH) content, which was restored by U0126, treatments with GSH-ethyl ester and dl-buthionine-(S,R)-sulfoximine revealed that GSH does not regulate cisplatin toxicity in the present experimental conditions. In spite of it, PD98059 and U0126 reduced the intracellular accumulation of cisplatin. These results suggest that GSH-independent modulation of drug transport is a major mechanism explaining the anti-apoptotic action of MEK/ERK inhibitors in cisplatin-treated myeloid cells.  (+info)

Kinetic evaluation of polyamines as radical scavengers. (35/170)

To clarify whether polyamines scavenge alkyl (carbon-centered) and peroxy (oxygen-centered) radicals, we analyzed their effects on the kinetics of polymerization of methyl methacrylate (MMA) induced by 2,2'-azobisisobutyronitrile (AIBN, a R* radical) and benzoyl peroxide (BPO, a PhCOO* radical) under nearly anaerobic conditions. Stoichiometric factors (n; number of free radicals trapped by one mole of antioxidant moiety) were determined by the induction period method. The n value for polyamines (putrescine, spermidine and spermine) was 0.1-0.7, whereas that for conventional synthetic antioxidants, BHA and BHT, was about 2. These n values were not different between the AIBN and BPO systems. The n value for polyamines declined in the order spermine > spermidine > putrescine. The K(inh)/K(p) value for polyamines (20-115) was greater than that (4-7) for BHT or BHA. Radical-scavenging activity largely depends on the stoichiometric factor of antioxidants rather than their effects on initial rate of polymerization, a rate of propagation. Polyamines may scavenge alkyl or peroxy radicals derived from polyunsaturated fatty acids in biological systems.  (+info)

Filarial glutathione S-transferase: its induction by xenobiotics and potential as drug target. (36/170)

Glutathione-S-transferase (GST) a Phase-II drug detoxification enzyme, was detected in Setaria cervi, a bovine filarial parasite. In vitro effect of diethylcarbamazine, butylated hydroxyanisole and phenobarbitone on the GST of adult female S. cervi was assayed by the addition of these compounds in the maintenance medium. The specific activity of GST towards 1-chloro-2,4-dinitrobenzene was increased progressively 1.2-1.97, 1.3-2.4 and 1.2-2.7 times at 10-100 microM of diethylcarbamazine, butylated hydroxyanisole and phenobarbitone, respectively, after 5 h at 37 degrees C. Substrate specificity studies showed a higher increase in specific activity with ethacrynic acid and no change with cumene hydroperoxide. Although the intensity of GST activity band was more in extract from diethylcarbamazine or butylated hydroxyanisole treated worms extract, an extra band of activity appeared in those worm extracts compared to control worm extract. SDS/PAGE showed increased thickness of the band corresponding to purified GST in extracts from diethylcarbamazine/butylated hydroxyanisole/phenobarbitone treated worms. Purification and quantification of GST from diethylcarbamazine and butylated hydroxyanisole treated worms indicated an increase in enzyme specific activity. The increase in GST protein by these agents was blocked by prior treatment with actinomycin D, indicative of a transcription dependent response. The role of this enzyme in motility and viability of microfilariae and adult female was tested in vitro using a range of known GST inhibitors. Of those tested, ethacrynic acid, ellagic acid, 1-chloro-2,4-dinitrobenzene, cibacron blue and butylated hydroxyanisole reduced the viability and motility of microfilariae and adult female worms at micromolar concentrations. These results suggest that S. cervi GST is inducible in response to the antifilarial drug diethylcarbamazine and may play an important role in parasite's survival, thus could be a potential drug target.  (+info)

Effects of 2(3)-tert-butyl-4-hydroxyanisole (BHA) on in situ mitochondria of Trypanosoma cruzi. (37/170)

Results obtained with in situ mitochondria of Trypanosoma cruzi showed that this protozoon had only two energy coupling sites, sites II and III that correspond to higher eukaryote mitochondria. Rotenone did not inhibit the oxygen uptake of the parasite. These results suggest that the NADH-ubiquinone segment of the respiratory chain has no activity. Studies with in situ mitochondria confirmed that BHA, an antioxidant food additive, blocks the mitochondrial electron transport chain at the succinate-cytochrome b segment being the molecular basis of this trypanocidal action.  (+info)

Determination and confirmation of five phenolic antioxidants in foods by LC/MS and GC/MS. (38/170)

Identification and determination of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) by means of LC/MS and GC/MS were examined. These five phenolic antioxidants were detected as their pseudo-molecular ions [M-H]- by LC/MS using a Shim-pack FC-ODS column with drying gas. Moreover, BHA, BHT and TBHQ were detected based on their mass fragment ions by GC/MS. Decomposition of TBHQ, NDGA and PG during analysis could be prevented by the addition of L-ascorbic acid (AsA) to the extraction solvent. All five antioxidants were extracted from nikuman, olive oils, peanut butter, pasta sauce and chewing gum with a mixture of acetonitrile-2-propanol-ethanol (2:1:1) containing 0.1% AsA (AsA mixture), which had been cooled in a freezer and filtered. One part filtrate and 5 parts water were mixed and placed on a Mega-Bond Elut C18 cartridge, except in the case of chewing gum. Lipids in foods were removed on a C18 cartridge by washing with 5 mL of 5% acetic acid, and antioxidants were eluted with 5 mL of AsA mixture. The antioxidants spiked into nikuman, olive oil, peanut butter, pasta sauce and chewing gum were successfully identified and their concentrations determined by LC/MS, and GC/MS with good recoveries.  (+info)

Kinetic studies of the radical-scavenging activity of ebselen, a seleno-organic compound. (39/170)

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is now under active investigation as a neuroprotective and anticancer agent. In the present study, the induction period method was used to investigate the antioxidant activity of ebselen in the radical polymerization of methyl methacrylate (MMA) at 70 degrees C. The reaction of ebselen with growing MMA radicals (lipid radicals) showed a k(inh) of 4 x 10(4) M(-1)s(-1). This value was similar to that for mercaptomethylimidazole (MMI, a thiol) and 10-fold greater than that for butylated hydroxyanisole (BHA). The ratio of the rate of chain inhibition to that of chain propagation (CI/CP) for ebselen, MMI and BHA was 0.1, 0.01 and 0.001, respectively, whereas the stoichiometric factor (n, the number of free radicals trapped by one mole of antioxidant moiety) for the corresponding compounds was 0.02, 0.2 and 2, respectively. Ebselen preferentially affected CP rather than CI, indicating that it was an effective scavenger (suppressor) of growing MMA radicals. These results suggest that ebselen is a potent suppressor of polyunsaturated fatty acid (PUFA) radicals, which are harmful radicals in biological systems.  (+info)

Methamphetamine-induced dopaminergic neurotoxicity is regulated by quinone-formation-related molecules. (40/170)

Recently, the neurotoxicity of dopamine (DA) quinone formation by auto-oxidation of DA has focused on dopaminergic neuron-specific oxidative stress. In the present study, we examined DA quinone formation in methamphetamine (METH)-induced dopaminergic neuronal cell death using METH-treated dopaminergic cultured CATH.a cells and METH-injected mouse brain. In CATH.a cells, METH treatment dose-dependently increased the levels of quinoprotein (protein-bound quinone) and the expression of quinone reductase in parallel with neurotoxicity. A similar increase in quinoprotein levels was seen in the striatum of METH (4 mg/kg X4, i.p., 2 h interval)-injected BALB/c mice, coinciding with reduction of DA transporters. Furthermore, pretreatment of CATH.a cells with quinone reductase inducer, butylated hydroxyanisole, significantly and dose-dependently blocked METH-induced elevation of quinoprotein, and ameliorated METH-induced cell death. We also showed the protective effect of tyrosinase, which rapidly oxidizes DA and DA quinone to form stable melanin, against METH-induced dopaminergic neurotoxicity in vitro and in vivo using tyrosinase null mice. Our results indicate that DA quinone formation plays an important role, as a dopaminergic neuron-specific neurotoxic factor, in METH-induced neurotoxicity, which is regulated by quinone formation-related molecules.  (+info)