Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

Lipid peroxidation products have a high reactivity against the primary amino groups of biomolecules such as aminophospholipids, proteins, and DNA. Until now, many papers have reported about the modification of biomolecules derived from lipid peroxides. Our group has also reported that aminophospholipids, such as phosphatidylethanolamine (PE), can be modified by lipid peroxidation including 13-hydroperoxyoctadecadienoic acid (13-HPODE). The aim of this study was to examine the oxidative stress in vivo by detecting the formation of N-(hexanoyl)phosphatidylethanolamine (HEPE) and N-(hexanoyl)phosphatidylserine (HEPS), a novel hexanoyl adduct, using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) and a monoclonal antibody.Consequently, we observed that the formation of HEPE and HEPS occurred in the red blood cell (RBC) ghosts modified by 13-HPODE and the oxidative stress model induced by carbon tetrachloride (CCl₄) using LC/MS/MS monitoring hexanoyl ethanolamine (HEEA), a head group of HEPE, and hexanoyl serine (HESE) as a part of HEPS. Furthermore, we obtained a novel type of monoclonal antibody against HEPE. This antibody could recognize HEPE in the liver of rats with oxidative stress in vivo.     

Nuclear factor erythroid 2-related factor-2 activity controls 4-hydroxynonenal metabolism and activity in prostate cancer cells

4-Hydroxynonenal (HNE) is an end product of lipoperoxidation with antiproliferative and proapoptotic properties in various tumors. Here we report a greater sensitivity to HNE in PC3 and LNCaP cells compared to DU145 cells. In contrast to PC3 and LNCaP cells, HNE-treated DU145 cells showed a smaller reduction in growth and did not undergo apoptosis. In DU145 cells, HNE did not induce ROS production and DNA damage and generated a lower amount of HNE-protein adducts. DU145 cells had a greater GSH and GST A4 content and GSH/GST-mediated HNE detoxification. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a regulator of the antioxidant response. Nrf2 protein content and nuclear accumulation were higher in DU145 cells compared to PC3 and LNCaP cells, whereas the expression of KEAP1, the main negative regulator of Nrf2 activity, was lower. Inhibition of Nrf2 expression with specific siRNA resulted in a reduction in GST A4 expression and GS-HNE formation, indicating that Nrf2 controls HNE metabolism. In addition, Nrf2 knockdown sensitized DU145 cells to HNE-mediated antiproliferative and proapoptotic activity. In conclusion, we demonstrated that increased Nrf2 activity resulted in a reduction in HNE sensitivity in prostate cancer cells, suggesting a potential mechanism of resistance to pro-oxidant therapy.     

Kuwanon W,a natural Diels—Alder type adduct from the root bark of Morus lhou

A natural Diels—Alder type adduct, named kuwanon W, was isolated from ethyl acetate extracts of the root bark of cultivated mulberry tree, and its structure was determined on the basis of spectral and chemical evidence. Kuwanon W is regarded biogenetically as a Diels—Alder adduct of a chalcone derivative and dehydroprenyl-flavone.     

Inhibitors of topoisomerase II enzymes: a unique group of environmental mutagens and carcinogens

Many inhibitors of topoisomerase II enzymes are potent mutagens, leading to major chromosomal deletions, illegitimate recombination and aneuploidy. There is increasing evidence that they are also human carcinogens. However, their lack of chemical reactivity means that they may give weak or negative results in commonly used mutagenicity tests, or may give data with characteristics quite distinct from chemicals that alkylate DNA. They do not form DNA adducts and assays such as ³²P-postlabelling will not detect their presence in the body. They are generally not point mutagens and may fail to provide distinctive fingerprints in mutation spectra. These characteristics may be limiting a realistic evaluation of their role in human carcinogenesis using current methodologies.     

In vivo detection of a novel endogenous etheno–DNA adduct derived from arachidonic acid and the effects of antioxidants on its formation

Previous studies showed that 7-(1′,2′-dihydroxyheptyl)-substituted etheno DNA adducts are products of reactions with the epoxide of (E)-4-hydroxy-2-nonenal, an oxidation product of ω-6 polyunsaturated fatty acids (PUFAs). In this work, we report the detection of 7-(1′,2′-dihydroxyheptyl)-1,N⁶-ethenodeoxyadenosine (DHHedA) in rodent and human tissues by two independent methods: a ³²P-postlabeling/HPLC method and an isotope dilution liquid chromatography–electrospray ionization–tandem mass spectrometry method, demonstrating for the first time that DHHedA is a background DNA lesion in vivo. We showed that DHHedA can be formed upon incubation of arachidonic acid with deoxyadenosine, supporting the notion that ω-6 PUFAs are the endogenous source of DHHedA formation. Because cyclic adducts are derived from the oxidation of PUFAs, we subsequently examined the effects of antioxidants, α-lipoic acid, Polyphenon E, and vitamin E, on the formation of DHHedA and γ-hydroxy-1,N²-propanodeoxyguanosine (γ-OHPdG), a widely studied acrolein-derived adduct arising from oxidized PUFAs, in the livers of Long Evans Cinnamon (LEC) rats. LEC rats are afflicted with elevated lipid peroxidation and prone to the development of hepatocellular carcinomas. The results showed that although the survival of LEC rats was increased significantly by α-lipoic acid, none of the antioxidants inhibited the formation of DHHedA, and only Polyphenon E decreased the formation of γ-OHPdG. In contrast, vitamin E caused a significant increase in the formation of both γ-OHPdG and DHHedA in the livers of LEC rats.     

Tetrahydropapaveroline and salsolinol alter45Ca2+ efflux within perfused hippocampus of unrestrained rats

The kinetics of⁴⁵Ca²⁺ efflux were examined at circumscribed sites in the perfused hippocampus of the freely moving rat with either one of two tetrahydroisoquinoline (TIQ) products, tetrahydropapaveroline (THP) or salsolinol. Guide tubes for unilateral push-pull perfusion were implanted stereotaxically to rest just above sites within the dorsal hippocampus. Upon recovery from surgery, a tissue site in the hippocampus was prelabeled with 1.0 l of⁴⁵Ca²⁺ (2.0 Ci) injected through the indwelling guide tube. After 16–20 hr had elapsed, successive push-pull perfusions of the site were carried out with an artificial cerebrospinal fluid (CSF), at 5.0 min intervals and at a rate of 20 l/min, in order to obtain a control washout curve of declining radioactivity. On the fifth of a series of 5.0 min perfusions, either THP or salsolinol was added to the perfusion medium in a concentration of 10 or 100 ng/l. Then the hippocampal site was perfused again with control CSF for the collection of an additional three samples. Although THP in both of the test concentrations generally augmented the efflux of⁴⁵Ca²⁺, the temporal course and magnitude of the enhancement depended on the anatomical site of the perfusion. In the more rostral hippocampal planes of AP 3.0 and AP 4.0, THP caused a delayed efflux of the cation, after the perfusion of THP had been discontinued, in nearly half of the loci reactive to the TIQ. Similarly, salsolinol enhanced significantly the efflux of⁴⁵Ca²⁺ in a concentration-dependent manner during the interval of its perfusion within the hippocampal plane of AP 3.0. These results suggest that both THP and salsolinol can affect differentially the kinetics of⁴⁵Ca²⁺ efflux and that these differences are contingent on the circumscribed anatomical site of push-pull perfusion. It is envisaged that a part of the neurochemical effects of the two TIQs when acting centrally are mediated by membrane mechanisms involving Ca²⁺ transport, metabolism or other cellular activity of the cation.     

Effect of glucose-cysteine adduct as a cysteine prodrug in rats

Summary Effect of intraperitoneal administration (5 mmol/kg of body weight) of glucose- cysteine adduct (glc-cys) as a cysteine prodrug in rat tissues was studied. Cysteine levels in liver and kidney increased to 1.08 and 1.98mol per g or ml, respectively, at 2h after the administration. GSH levels did not change substantially. However, when glc-cys was injected to rats treated with diethyl maleate, a GSH-depleting agent, the decreased GSH levels were restored rapidly. The recoveries in liver and kidney were 72% at 1h and 66% at 2h, respectively, after glc-cys administration. Metabolism of glc-cys was assessed by urinary excretion of glc-cys, sulfate and taurine. Average excretion of glc-cys was 2.86mmol/kg/24h after glc-cys administration. Increased excretions of sulfate and taurine were 0.77 and 0.14mmol/kg/24h, respectively. Data show that, although glc-cys excretion was relatively rapid, glc-cys was effectively utilized for GSH synthesis in GSH-depleted tissues.