Peroxidase-catalyzed effects of indole-3-acetic acid and analogues on lipid membranes, DNA, and mammalian cells in vitro

This study aimed to explore the mechanisms and molecular parameters which control the cytotoxicity of derivatives of indole-3-acetic acid (IAA) when oxidatively activated by horseradish peroxidase (HRP). Lipid peroxidation was measured in liposomes, damage to supercoiled plasmid DNA assessed by gel electrophoresis, free radical intermediates detected by EPR following spin trapping, binding of IAA-derived products demonstrated by 3H labelling, stable products measured by HPLC, and cytotoxicity in hamster fibroblasts measured by clonogenic survival. IAA, and nine analogues more easily oxidized by HRP, caused lipid peroxidation in liposomes, but not detectably in membranes of hamster fibroblasts, and were cytotoxic after HRP activation to varying degrees. Cytotoxicity was not correlated with activation rate. The hydrophilic vitamin E analogue, Trolox, inhibited cytotoxicity, whereas loading fibroblasts with vitamin E was ineffective, consistent with an oxidative mechanism in which radical precursors to damage are intercepted by Trolox in the aqueous phase. However, two known oxidation products were nontoxic (the 3-carbinol and 3-aldehyde, both probably produced from 3-CH2OO* peroxyl radicals via the 3-CH*2 [skatolyl] radical following decarboxylation of the radical cation). The skatolyl radical from IAA was shown by EPR with spin trapping to react with DNA; electrophoresis showed binding to occur. Treatment of hamster fibroblasts with 5-3H-IAA/HRP resulted in intracellular bound 3H. Together with earlier results, the new data point to unknown electrophilic oxidation products, reactive towards intracellular targets, being involved in cytotoxicity of the IAA/HRP combination, rather than direct attack of free radicals, excited states, or membrane lipid peroxidation.     

The reaction of oxygen with radicals from oxidation of tryptophan and indole-3-acetic acid

The oxidation of tryptophan and indole-3-acetic acid (IAA) by the dibromine radical anion or peroxidase from horseradish in aqueous solution was investigated and compared, especially with respect to the involvement of oxygen and superoxide. Using EPR with spin-trapping, the tryptophanyl radical, generated by either method was found to react with oxygen, although this reaction is too slow to be observed by pulse radiolysis (k < 5 x 10(6) dm3 mol-1 s-1). No superoxide results from this reaction, thus excluding an electron-transfer mechanism and suggesting the formation of a tryptophan peroxyl radical, possibly in a reversible process. These observations imply that in proteins where the tryptophanyl radical exists as a stable species it must either have its reactivity modified by the protein environment or be inaccessible to oxygen. The related molecule LAA is oxidized by either peroxidase or Br2.- to a radical cation that decarboxylates to yield a skatolyl radical. The latter reacts with oxygen to give a peroxyl radical that does not release superoxide. However, O2.- is formed during the peroxidase-catalyzed oxidation of indoleacetic acid. This supports the hypothesis that the peroxidase can act in an oxidase cycle involving ferrous enzyme and compound III, with superoxide as a product.     

N-terminal 4-imidazolidinone prodrugs of Leu-enkephalin: synthesis, chemical and enzymatic stability studies

Four N-terminal 4-imidazolidinone prodrugs of Leu-enkephalin are prepared and characterized. Their enzymatic and chemical stability are assessed using high-performance liquid chromatography. The prodrug derivatives are shown to degrade stoichiometrically to Leu-enkephalin in phosphate buffer [t1/2 (0.05 M phosphate buffer without KCl): acetone prodrug (II) 930 min; cyclopentanone prodrug (III): 216 min; cyclohexanone prodrug (IV): 432 min; 4-methylcyclohexanone prodrug (V): 792 min]. Furthermore, the prodrugs are shown to afford global stabilization of the Leu-enkephalin molecule towards the enzymes, aminopeptidase N and angiotensin converting enzyme, primarily responsible for degradation of Leu-enkephalin at the blood-brain barrier and in plasma. Therefore, the 4-imidazolidinones, being metabolic stable and bioreversible, may be suitable prodrug candidates for delivery of Leu-enkephalin to important target areas such as the brain, if given intravenously.     

The hypothalamic-pituitary axis: co-development of two organs

The C-525 laser dye at micromolar concentration range is shown to enhance up to two to three orders of magnitude the chemiluminescence (CL) accompanying tert-butyl hydroperoxide (t-BHP)-induced rat liver microsome oxidation and Fe(2+)-induced lipid peroxidation (LPO) in liposomes. C-525 is shown to be a more efficient sensitizer of CL accompanying LPO in membrane systems than the known low-energy excited triplet carbonyl sensitizer, chlorophyll-alpha, (Cl-a). Regarding the sensitization mechanism, C-525 and Cl-a were compared in (a) a peroxyl radical-producing system (2,2'-azobis(2-dimethylvaleronitrile) (AMVN); (b) excited carbonyl-producing systems (3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD) thermal decomposition and horseradish peroxidase (HRP)-catalyzed isobutanal oxidation); and (c) excited singlet oxygen-producing system [endoperoxide of 3,3-(1,4-naphthylidene)-dipropionate (NDPO2)]. C-525 sensitized CL only in the systems where peroxyl radical and/or triplet excited carbonyls are produced, the mechanism of CL sensitization apparently is energy transfer from the excited triplet carbonyls formed in the peroxyl radical self-reaction via Russell's mechanism or by dioxetane decomposition. Cl-a was found to considerably sensitize CL related to NDPO2 thermal decomposition, a source of singlet oxygen, in addition to acting as a sensitizer of triplet carbonyl CL. The chemical stability of the C-525 laser dye in excited state-generating systems was shown to be appropriate for its application as a sensitizer of CL related to LPO reactions in membranes, but not in the HRP-catalyzed peroxidation system.     

Alterations of antioxidant enzymes and oxidative damage to macromolecules in different organs of rats during aging

Oxygen free radicals have been hypothesized to play an important role in the aging process. To investigate the correlation between the oxidative stress and aging, we have determined the levels of oxidative protein damage and lipid peroxidation in the brain and liver, and activities of antioxidant enzymes in the brain, liver, heart, kidney, and serum from the Fisher 344 rats at ages of 1, 6, 12, 18, and 24 months. The results showed that the level of oxidative protein damage (measured as carbonyl content) in the brain and liver was significantly higher in older animals than in young animals. No statistical difference was observed in the lipid peroxidation of the liver and brain between young and old animals. The activities of antioxidant enzymes in most tissues displayed an age-dependent decline. Superoxide dismutases in the heart, kidney, and serum, glutathione peroxidase activities in the serum and kidney, and catalase activities in the brain, liver, and kidney, significantly decreased during aging. Cytochrome c oxidase, an enzyme involved in electron transport in mitochondria, initially increased, but subsequently decreased in the aged brain, whereas no significant alteration was observed in the liver mitochondrial antioxidant enzymes. The present studies suggest that the accumulation of oxidized proteins during aging is most likely to be linked with an age-related decline of antioxidant enzyme activities, whereas lipid peroxidation is less sensitive to predict the aging process.     

Combination gene delivery of the cell cycle inhibitor p27 with thymidine kinase enhances prodrug cytotoxicity

Cytoxicity induced by the herpesvirus thymidine kinase (TK) gene in combination with prodrugs is dependent on cell growth and leads to the elimination of genetically modified cells, thus limiting the duration of expression and efficacy of this treatment in vivo. Here, an effort was made to enhance TK/prodrug efficacy by coexpression of a cyclin-dependent kinase inhibitor (CKI), p27, to render cells resistant to TK/prodrug by inhibiting DNA synthesis. Expression of p27 by transfection substantially reduced cell cycle progression, and its activity was enhanced by mutations designed to stabilize the protein. Coexpression of p27 and TK or a p27/TK fusion protein led to greater prodrug cytotoxicity than that produced by TK alone in the Renca cell line, which is sensitive to bystander killing. Combination gene transfer of this CKI with TK therefore sustained the synthesis of TK by genetically modified cells to enhance the susceptibility of bystander cells to prodrug cytotoxicity and increased the efficacy of this gene transfer approach.     

The choice of prodrugs for gene directed enzyme prodrug therapy of cancer

Prodrugs are chemicals that are pharmacodynamically and toxicologically inert but which can be converted to highly active species. In cancer chemotherapy, enzyme activated prodrugs have been effective against certain animal tumours. However, in the clinic it has been found that human tumours containing appropriately high levels of the activating enzymes were rare and not associated with any particular type of tumour. Gene directed enzyme prodrug therapy (GDEPT) attempts to overcome this problem by killing tumour cells by the activation of a prodrug after the gene encoding for an activating enzyme has been targeted to the malignant cell. Here we summarise the various enzyme/prodrug systems that have been proposed for cancer therapy and comment on their suitability for GDEPT. This is because systems developed for other applications such as antibody directed enzyme prodrug therapy (ADEPT) may not be suitable for GDEPT. What is required are nontoxic prodrugs that can be converted intracellularly to highly cytotoxic metabolites that are not cell cycle specific in their mechanism of action. The active drugs released should also be readily diffusible and exert a bystander effect. Alkylating agents best meet these criteria. An example of a suitable enzyme/prodrug system may be a bacterial nitroreductase that can convert a relatively nontoxic monofunctional alkylating agent to a difunctional alkylating agent that is some ten thousand times more cytotoxic.     

Random versus selective membrane phospholipid oxidation in apoptosis: role of phosphatidylserine

The formation of reactive oxygen species has been associated with apoptosis. To assess the role of lipid peroxidation in apoptosis, we used 2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN) to generate peroxyl radicals within cellular membranes of HL-60 cells. cis-Parinaric acid (cis-PnA) metabolically integrated into phospholipids of HL-60 cells was used as a probe to assess the extent of lipid peroxidation within specific phospholipid classes. Within 2 h, AMVN (500 microM) randomly oxidized more than 85% of cis-PnA contained in all major classes of phospholipids. AMVN-induced lipid peroxidation was followed by apoptosis as determined by nuclear condensation, DNA fragmentation, and annexin V binding to externalized phosphatidylserine (PS). Fluorescamine derivatization of external aminophospholipids revealed that PS, but not phosphatidylethanolamine, was externalized. The vitamin E analogue, 6-hydroxy-2,2,5,7,8-pentamethylchromane (PMC), inhibited overall oxidation of cis-PnA in phospholipids by more than 85%. Not all phospholipids, however, were equally protected. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin were nearly completely protected by PMC, while oxidation of PS was unaffected in whole living cells. The insensitivity of PS to PMC was not an intrinsic property because PMC protected all lipids equally during AMVN oxidation of liposomes prepared from cis-PnA-labeled cells. The potential role for PS oxidation in apoptosis was further suggested by the faithful execution of apoptosis following coexposure of cells to AMVN and PMC.     

Extracellular matrix components reflect the dynamics of a healing tympanic membrane perforation--a histochemical study

Peroxidation of unsaturated fatty acids in membrane phospholipids is one of the multiple cytotoxic effects of oxidative stress. Lipid peroxidation is significant because a single initiating event triggers a chain reaction, thus amplifying the initial stimulus. Many oxidative stress-related pathologies have been linked to lipid peroxidation. Mouse glutathione S-transferase mGSTA4-4 exhibits high glutathione conjugating activity with toxic products of lipid peroxidation, e.g., 4-hydroxynon-2-enal. In addition, mGSTA4-4 has glutathione peroxidase activity toward phospholipid hydroperoxides. On the basis of these catalytic properties, we have previously proposed that the enzyme may be physiologically important in alleviating the cytotoxic effects of lipid peroxidation. We have now experimentally confirmed this hypothesis by transfecting HepG2 cells with mGSTA4 cDNA, and demonstrating a protective effect of expressed mGSTA4-4 protein on cells exposed during plating to H2O2, organic hydroperoxides, and phosphatidylcholine hydroperoxide. As compared to cells transfected with insert-free vector, a larger proportion of mGSTA4-transfected cells was able to attach to the culture dish, and continued to divide in the presence of the above compounds. In addition to alleviating the cytotoxic effects of oxidative stress, mGSTA4-4 may interfere with the subtoxic but cytostatic signals generated by a low-level pro-oxidant state.     

Role of apolipoprotein B-derived radical and alpha-tocopheroxyl radical in peroxidase-dependent oxidation of low density lipoprotein

The peroxidation of low density lipoprotein (LDL) may play an important role in the modification of the lipoprotein to an atherogenic form. The oxidation of LDL by peroxidases has recently been suggested as a model for in vivo transition metal ion-independent oxidation of LDL (Wieland, E., S. Parthasarathy, and D. Steinberg. 1993. Proc. Natl. Acad. Sci. USA. 90: 5929-5933). It is possible that in vivo the peroxidase activities of proteins, such as prostaglandin synthase and myeloperoxidase, promote LDL oxidation. We have used horseradish peroxidase (HRP) and H2O2 as a model of peroxidase-dependent oxidation of LDL and we observed the following during HRP/H2O2-initiated LDL oxidation. i) The oxidation of alpha-tocopherol occurred with the concomitant formation of alpha-tocopheroxyl radical. This was followed by the production of an apolipoprotein B (apoB)-derived radical. The apoB radical and the alpha-tocopheroxyl radical were formed under both aerobic and anaerobic conditions. ii) Inclusion of N-t-butyl-alpha-phenylnitrone (PBN) did not inhibit alpha-tocopheroxyl radical formation. The ESR spectrum of a PBN/LDL-lipid derived adduct was observed after prolonged incubation. iii) There was formation of conjugated dienes, lipid hydroperoxides and thiobarbituric acid reactive substances. Our data indicate that HRP/H2O2 oxidizes both alpha-tocopherol and apoB to the corresponding radicals and concomitantly initiates lipid peroxidation.     

O6-alkylguanine-DNA alkyltransferase inactivation by ester prodrugs of O6-benzylguanine derivatives and their rate of hydrolysis by cellular esterases

To modulate the bioavailability and perhaps improve the tumor cell selectivity of O6-alkylguanine-DNA alkyltransferase (AGT) inactivators, pivaloyloxymethyl ester derivatives of O6-benzylguanine (BG) were synthesized and tested as AGT inactivators and as substrates for cellular esterases. The potential prodrugs examined were the 7- and 9-pivaloyloxymethyl derivatives of O6-benzylguanine (7- and 9-esterBG), and of 8-aza-O6-benzylguanine (8-aza-7-esterBG and 8-aza-9-esterBG) and the 9-pivaloyloxymethyl derivative of 8-bromo-O6-benzylguanine (8-bromo-9-esterBG). The benzylated purines were all potent inactivators of the pure AGT and of the AGT activity in HT29 cells and cell extracts. Each ester was at least 75 times less potent than the corresponding benzylated purine against the pure human AGT. In contrast, the activities of esters and their respective benzylated purine were similar in crude cell extracts and in intact cells. The increase in potency of esters in cellular extracts could be explained by a conversion of the respective prodrug to the more potent benzylated purine in the presence of cellular esterases. The apparent catalytic activity (Vmax/Km) of liver microsomal esterase for 8-azaBG ester prodrugs was 70-130 times greater than for BG prodrugs and 10-20 times greater than for 8-bromo-9-esterBG. Tumor cell hydrolysis of the esters varied considerably as a function of cell type and prodrug structure. These data suggest that these or related prodrugs may be advantageous for selective AGT inactivation in certain tumor types.     

Hantavirus antigen detection in kidney biopsies from patients with nephropathia epidemica

We investigated the inhibition mechanism of lipid peroxidation by estrogens. Estradiol and 2-hydroxyestradiol showed strong inhibitory activities toward NADPH and ADP-Fe(3+)-dependent lipid peroxidations in the microsomes from rat livers only when the steroids were added to the reaction system before the start of the peroxidation reaction. These steroids also strongly inhibited oxygen uptake only when added before the start of the reaction. These results suggest that estradiol and 2-hydroxyestradiol inhibit the initial stage of microsomal lipid peroxidation. Lipid peroxidation of erythrocyte membranes induced by the systems of xanthine oxidase-hypoxanthine and ascorbate was strongly inhibited by 2-hydroxyestradiol, but not by estradiol. Lipid peroxidation of erythrocyte membranes induced by 2.2'-azobis- (amidinopropane) dihydrochloride was not markedly inhibited by estradiol and 2-hydroxyestradiol, suggesting that the steroids have low reactivity with lipid peroxyl radicals. However, lipid peroxidation induced by t-butyl hydroperoxide-Fe3+ was strongly inhibited only by 2-hydroxyestradiol. It seems that 2-hydroxyestradiol may interact with alkoxyl rather than with peroxyl radicals during lipid peroxidation.     

Pacemaker event counters: possible sources of error in calculation of AV synchrony in VDD single lead systems as an example for present limitations

1. The in vitro effects of alloxan, dialuric acid and vanadium ions, alone or in combination, on lipid peroxidation and on antioxidant enzyme activity in rat liver and kidney were studied. 2. Unlike alloxan, alloxan-glutathione (GSH) and dialuric acid increased lipid peroxidation, which could be explained by the decreased activity of catalase and GSH peroxidase during incubation. 3. Vanadium(IV) ions increased the amount of thiobarbituric acid-reacting substances, but neither vanadium(IV) nor vanadium(V) changed the enzyme activity. 4. The combination of vanadium ions and alloxan-GSH or dialuric acid had no additive effect on lipid peroxidation. Vanadium ions decreased the dialuric acid-induced inhibition of catalase activity. 5. The present results suggest the therapeutic value of vanadium as an antidiabetic agent.     

The involvement of cytochrome P450 peroxidase in the metabolic bioactivation of cumene hydroperoxide by isolated rat hepatocytes

Organic hydroperoxides are believed to be primarily detoxified in cells by the GSH peroxidase/GSSG reductase system and activated to cytotoxic radical species by non-heme iron. However, organic hydroperoxides seem to be bioactivated by cytochrome P450 (P450) in isolated hepatocytes as various P450 (particularly P450 2E1) inhibitors inhibited cumene hydroperoxide (CumOOH) metabolism and attenuated subsequent cytotoxic effects including antimycin A-resistant respiration, lipid peroxidation, iron mobilization, ATP depletion, and cell membrane disruption. CumOOH metabolism was also faster in P450 1A-induced hepatocytes and was inhibited by the P450 1A inhibitor alpha-naphthoflavone. The ferric chelator deferoxamine also prevented cytotoxicity even after CumOOH had been metabolized but had no effect on CumOOH metabolism. This emphasizes the toxicological significance of the iron released following hydroperoxide metabolic activation by cytochrome P450. The radical trap, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), had no effect on CumOOH metabolism but prevented CumOOH-induced antimycin A-resistant respiration, lipid peroxidation, iron mobilization, and loss of membrane integrity. These results suggest that CumOOH is metabolically activated by some P450 enzymes (e.g., P450 2E1) in hepatocytes to form reactive radical metabolites or oxidants that cause lipid peroxidation and cytotoxicity.     

New prodrug activation gene therapy for cancer using cytochrome P450 4B1 and 2-aminoanthracene/4-ipomeanol

Vector-mediated transfer of prodrug-activating genes provides a promising means of cancer gene therapy. In a search for more selective and more potent bioactivating enzymes for gene therapy of malignant brain tumors, the toxicity-generating capacity of the rabbit cytochrome P450 isozyme CYP4B1 was investigated. Rabbit CYP4B1, but not rat or human isozymes, efficiently converts the inert prodrugs, 2-aminoanthracene (2-AA) and 4-ipomeanol (4-IM), into highly toxic alkylating metabolites. Toxicity of these two prodrugs was evaluated in culture in parental and genetically modified rodent (9L) and human (U87) glioma cell lines stably expressing CYP4B1, and in vivo in a subcutaneous 9L tumor model in nude mice. The most sensitive CYP4B1-expressing glioma clone, 9L4B1-60, displayed an LD50 of 2.5 microM for 2-AA and 4-IM after 48 h of prodrug incubation, whereas 20 times higher prodrug concentrations did not cause any significant toxicity to control cells. Substantial killing of control tumor cells by 2-AA was achieved by co-culturing these cells with CYP4B1-expressing cells at a ratio of 100:1, and toxic metabolites could be transferred through medium. In both CYP4B1-expressing cells and co-cultured control cells, prodrug bioactivation was associated with DNA fragmentation, as assayed by fluorescent TUNEL assays and by annexin V staining. Alkaline elution of cellular DNA after exposure to 4-IM revealed extensive protein-DNA crosslinking with single-strand breakage. Growth of 9L-4B1 tumors in nude mice was inhibited by intraperitoneal injection of 4-IM with minimal side effects. Potential advantages of the CYP4B1 gene therapy paradigm include: the low concentrations of prodrug needed to kill sensitized tumor cells; low prodrug conversion by human isozymes, thus reducing toxicity to normal cells; a tumor-killing bystander effect that can occur even without cell-to-cell contact; and the utilization of lipophilic prodrugs that can penetrate the blood-brain barrier.     

Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-alpha-phenylalanine). Thus, the kcat/Km value for the wild type hCPA1 with MTX-alpha-phenylalanine is 0.44 microM-1 s-1, and kcat/Km values for hCPA1-T268G with MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine are 1.8 and 0.16 microM-1 s-1, respectively. The cytotoxic efficiency of hCPA1-268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with the in vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.     

Novel fluorescein-based flow-cytometric method for detection of lipid peroxidation

The novel property of fluorescein to detect peroxyl radicals is demonstrated. On the basis of this observation, a fluorescein-based, flow-cytometric method to directly and continuously detect free radicals generated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein-labeled polylysine lose their fluorescence gradually upon addition of a peroxyl-radical-generating system (thermal decomposition of 2,2'-azobis(2-amidinopropane) [AAPH]). 5-/6-CF retains its fluorescence when exposed to AAPH in the presence of the peroxyl radical scavenger Trolox. When 5-/6-CF free in solution is incubated with red blood cells exposed to cumene hydroperoxide (CH), a similar loss of fluorescence occurs due to lipid peroxidation on RBC membranes, which is preventable by pretreatment of the cells with Trolox or vitamin E. Undecylamine-fluorescein (C11-fluor), a lipophilic fluorescein conjugate, has been incorporated into the membranes of RBC. Upon addition of CH, a decrease in fluorescence is fluorometrically observed that is proportional to the amount of hydroperoxide added and inhibited by preincubation with Trolox or vitamin E. Flow-cytometric studies are then performed to demonstrate that C11-fluor can monitor free radicals generated during lipid peroxidation on a cell-by-cell basis. When exposed to CH, a time-dependent shift of the flow-cytometric profile toward lower values is observed that is inhibited by Trolox or vitamin E. This approach in conjunction with multiparametric flow cytometry may allow examination of the biologic significance of lipid peroxidation by correlation to other cellular end points on single cells.     

Effects of diltiazem on myocardial perfusion abnormalities during exercise in patients with hypertrophic cardiomyopathy

We developed a novel chemiluminescent assay of beta-D-galactosidase (beta-gal) based on the chemiluminescence of indole. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) was used as a substrate for beta-gal and also as a light emitter. X-gal was hydrolysed by beta-gal to liberate free indoxyl, followed by oxidation to indigo dye, and simultaneously produces hydrogen peroxide (H2O2). H2O2 reacts with the residual X-gal in the presence of horseradish peroxidase (HRP) to emit light. The measurable range of beta-gal obtained by this method was 6 x 10(-14) mol/L to 6 x 10(-11) mol/L; the detection limit was 3 amol/assay. This chemiluminescent assay could be applied to an enzyme immunoassay of thyroxine using beta-gal as the enzyme label.     

gamma-Glutamyl transpeptidase-dependent lipid peroxidation in isolated hepatocytes and HepG2 hepatoma cells

Gamma-glutamyltranspeptidase (GGT), a plasma membrane-bound enzyme, provides the only activity capable to effect the hydrolysis of extracellular glutathione (GSH), thus favoring the cellular utilization of its constituent amino acids. Recent studies have shown however that in the presence of chelated iron prooxidant species can be originated during GGT-mediated metabolism of GSH, and that a process of lipid peroxidation can be started eventually in suitable lipid substrates. The present study was undertaken to verify if a GGT-dependent lipid peroxidation process can be induced in the lipids of biological membranes, including living cells, and if this effect can be sustained by the GGT highly expressed at the surface of HepG2 human hepatoma cells. In rat liver microsomes (chosen as model membrane lipid substrate) exposed to GSH and ADP-chelated iron, the addition of GGT caused a marked stimulation of lipid peroxidation, which was further enhanced by the addition of the GGT co-substrate glycyl-glycine. The same was observed in primary cultures of isolated rat hepatocytes, where the lipid peroxidation process did not induce acute toxic effects. GGT-stimulation of lipid peroxidation was dependent both on the concentration of GSH and of ADP-chelated iron. In GGT-rich HepG2 human hepatoma cells, the exposure to GSH, glycyl-glycine, and ADP-chelated iron resulted in a nontoxic lipid peroxidation process, which could be prevented by means of GGT inhibitors such as acivicin and the serine-boric acid complex. In addition, by co-incubation of HepG2 cells with rat liver microsomes, it was observed that the GGT owned by HepG2 cells can act extracellularly, as a stimulant on the GSH- and iron-dependent lipid peroxidation of microsomes. The data reported indicate that the lipid peroxidation of liver microsomes and of living cells can be stimulated by the GGT-mediated metabolism of GSH. Due to the well established interactions of lipid peroxidation products with cell proliferation, the phenomenon may bear particular significance in the carcinogenic process, where a relationship between the expression of GGT and tumor progression has been envisaged.