Reaction of uracil with hypochlorous acid.

The end products of the reaction of uracil with at least a 10-fold excess of aqueous hypochlorous acid at pH 7–8 were found to be trichloroacetic acid, carbon dioxide and nitrogen trichloride. Little formation of trichloroacetic acid was observed after 24 hours when the ratio of hypochlorous acid to uracil was less than 4:1. An intermediate in the reaction was found to be 5-chlorouracil. This was also degraded by hypochlorous acid to trichloroacetic acid.     

Biomarkers of myeloperoxidase-derived hypochlorous acid

Hypochlorous acid is the major strong oxidant generated by neutrophils. The heme enzyme myeloperoxidase catalyzes the production of hypochlorous acid from hydrogen peroxide and chloride. Although myeloperoxidase has been implicated in the tissue damage that occurs in numerous diseases that involve inflammatory cells, it has proven difficult to categorically demonstrate that it plays a crucial role in any pathology. This situation should soon be rectified with the advent of sensitive biomarkers for hypochlorous acid. In this review, we outline the advantages and limitations of chlorinated tyrosines, chlorohydrins, 5-chlorocytosine, protein carbonyls, antibodies that recognize HOCl-treated proteins, and glutathione sulfonamide as potential biomarkers of hypochlorous acid. Levels of 3-chlorotyrosine and 3,5-dichlorotyrosine are increased in proteins after exposure to low concentrations of hypochlorous acid and we conclude that their analysis by gas chromatography and mass spectrometry is currently the best method available for probing the involvement of oxidation by myeloperoxidase in the pathology of particular diseases. The appropriate use of other biomarkers should provide complementary information.Keywords-Free radicals, Myeloperoxidase, Neutrophil oxidant, Hypochlorous acid, Chlorotyrosine, Chlorohydrin, Oxidant biomarker     

Hydroxyurea: effects on deoxyribonucleotide pool sizes correlated with effects on DNA repair in mammalian cells

We have measured deoxyribonucleotide pool sizes in different cell types: normal human, transformed human (HeLa), and the permanent hamster line CHO-K1. The range of sizes of the four DNA precursor pools in CHO cells is far greater than in human cells. It is a general rule that hydroxyurea causes rapid depletion of pools (except for dTTP) until the pool present in smallest amount is exhausted; this suggests a tight coupling of the pools to DNA replication (the presumed main cause of the depletion). The effect of hydroxyurea on DNA repair after ultraviolet irradiation (namely, a relatively small accumulation of incomplete repair sites blocked at the resynthesis stage) is probably accounted for by the reduced availability of DNA precursors. However, depletion of the dCTP pool is not an adequate explanation for the observed enhancement by hydroxyurea of the inhibitory effect of cytosine arabinoside; we suggest other possible modes of action. Ultraviolet irradiation has only small effects on the levels of deoxyribonucleotides.     

Cryoprotective effects of D-allose on mammalian cells

D-allose, an aldo-hexose, is a rare sugar whose biological functions remain largely unclear. Recently, we demonstrated a novel inhibitory effect of D-allose on production of reactive oxygen species (ROS). Here, we focused on investigating cryoprotective effects of D-allose on cell viability. Mammalian cell lines including OVCAR-3 (human ovarian cancer), HeLa (human cervical cancer), HaCaT (human skin keratinocytes), HDF (human dermal fibroblasts) and NIH3T3 (murine fibroblasts) cells were frozen at -80 degrees C in culture media with various D-allose concentrations. Cells were allowed to recover for 24 h, 1 week or 1 month prior to survival assessment using the trypan blue dye exclusion test, when cell proliferation was evaluated by MTT assay. A beneficial protective role of D-allose on cell survival was found, similar to that of trehalose (disaccharide of glucose), a recognized cryoprotectant. The results suggest that D-allose as a sole additive may provide effective protection for mammalian cells during freezing. Practical studies now need to be performed with D-allose, for example to determine optimal freezing protocols and explore potential for preservation of tissues or organs at non-freezing temperatures.     

Induction of fatty acid synthesis in cultured mammalian cells: effects of cycloheximide and X-rays

It was found that L cells deprived of exogenous fatty acids responded adaptively by increased synthesis of fatty acids. This adaptive increase was found to be sensitive to cycloheximide presented at the time of removal of exogenous lipids, but was not sensitive to cycloheximide added six hours after removal of lipids. It was concluded that the adaptive increase in fatty acid synthesis represents a case of enzyme induction. The effect of x-rays on this process was studied. It was found that induction was inhibited to a greater extent than total protein synthesis. Factors of potential importance to such an effect are discussed.     

Modification of phosphatidylserine by hypochlorous acid

The binding of the heme enzyme myeloperoxidase to phosphatidylserine epitopes on the surface of non-vital polymorphonuclear leukocytes and other cells at inflammatory sites favours modifications of this phospholipid by myeloperoxidase products. As detected by MALDI-TOF mass spectrometry hypochlorous acid and the myeloperoxidase-hydrogen peroxide-chloride system convert 1,2-dipalmitoyl-sn-glycero-3-phosphoserine into 1,2-dipalmitoyl-sn-glycero-3-phosphoacetaldehyde and 1,2-dipalmitoyl-sn-glycero-3-phosphonitrile. A transient chlorimine derivative was detected using 4-chloro-α-cyanocinnamic acid as matrix in mass spectrometry only at short incubation times and supplying HOCl in two-fold excess. The decay of transient chlorinated products was followed by changes in absorbance spectra using O-phospho-l-serine to model the behavior of the serine head group in phosphatidylserine. N-Chlorimine and N-monochloramine derivatives decayed with half-life times of 1.5 and 57 min, respectively, at 22 °C and pH 7.4. N-Dichloramines decayed within few seconds under these conditions.     

The reactivity of myeloperoxidase compound I formed with hypochlorous acid

The reaction of human myeloperoxidase (MPO) with hypochlorous acid (HOCl) was investigated by conventional stopped-flow spectroscopy at pH 5, 7, and 9. In the reaction of MPO with HOCl, compound I is formed. Its formation is strongly dependent on pH. HOCl (rather than OCl-) reacts with the unprotonated enzyme in its ferric state. Apparent second-order rate constants were determined to be 8.1 x 10(7) M(-1)s(-1) (pH 5), 2.0 x 10(8) M(-1)s(-1) (pH 7) and 2.0 x 10(6) M(-1)s(-1) (pH 9) at 15 degrees C. Furthermore, the kinetics and spectra of the reactions of halides and thiocyanate and of physiologically relevant one-electron donors (ascorbate, nitrite, tyrosine and hydrogen peroxide) with this compound I were investigated using the sequential-mixing technique. The results show conclusively that the redox intermediates formed upon addition of either hydrogen peroxide or hypochlorous acid to native MPO exhibit the same spectral features and reactivities and thus are identical. In stopped-flow investigations, the MPO/HOCl system has some advantage since: (i) in contrast to H2O2, HOCl cannot function as a one-electron donor of compound I; and (ii) MPO can easily be prevented from cycling by addition of methionine as HOCl scavenger. As a consequence, the observed absorbance changes are bigger and errors in data analysis are smaller.     

Antineoplastic effects of natural killer cells sorted with flow cytometry on brain tumors

For the purpose of FCM, we have developed a new method of sterile sorting of NK cells by negative selection using FITC-labelled monoclonal antibodies. In this method, over 95% lymphocytes with large granular lymphocytes (NK cells) were obtained by exclusion of Leu-1 and HLA-DR positive cells from human peripheral blood. From the results of cytotoxicity test of NK cells against 4 types of glioma cell lines and 6 kinds of cultured cells from surgical materials, the antineoplastic effect was recognized in 4 out of 6 surgical materials. Administration of NK cells through an Ommaya tube in 2 brain tumor patients caused no side effects. This technique is clinically applicable because 1) NK cells are easy to obtain, 2) NK cells show antineoplastic effect on brain tumors, and 3) the sensitivity can be evaluated in individual patients.     

The reactivity of myeloperoxidase compound I formed with hypochlorous acid

Abstract

The reaction of human myeloperoxidase (MPO) with hypochlorous acid (HOCl) was investigated by conventional stopped-flow spectroscopy at pH 5, 7, and 9. In the reaction of MPO with HOCl, compound I is formed. Its formation is strongly dependent on pH. HOCl (rather than OCl^-) reacts with the unprotonated enzyme in its ferric state. Apparent second-order rate constants were determined to be 8.1×10⁷ M^-1s^-1 (pH 5), 2.0×10⁸ M^-1s^-1 (pH 7) and 2.0×10⁶ M^-1s^-1 (pH 9) at 15°C. Furthermore, the kinetics and spectra of the reactions of halides and thiocyanate and of physiologically relevant one-electron donors (ascorbate, nitrite, tyrosine and hydrogen peroxide) with this compound I were investigated using the sequential-mixing technique. The results show conclusively that the redox intermediates formed upon addition of either hydrogen peroxide or hypochlorous acid to native MPO exhibit the same spectral features and reactivities and thus are identical. In stopped-flow investigations, the MPO/HOCl system has some advantage since: (i) in contrast to H₂O₂, HOCl cannot function as a one-electron donor of compound I; and (ii) MPO can easily be prevented from cycling by addition of methionine as HOCl scavenger. As a consequence, the observed absorbance changes are bigger and errors in data analysis are smaller.     

Different effects of hypochlorous acid on human neutrophil metalloproteinases: activation of collagenase and inactivation of collagenase and gelatinase.

Human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) produce the reactive oxidant hypochlorous acid (HOCl) and release the matrix metalloproteinases collagenase and gelatinase from secretory granules. We have investigated the stoichiometry of activation and inactivation of the two metalloproteinases with HOCl. HOCl activated purified neutrophil procollagenase at ratios between 10 and 40 mol of HOCl/mol enzyme, but caused inactivation at higher ratios. Maximum activation was about the same as that achieved by p-aminophenyl-mercuric acetate. However, less than a third of the total collagenase released from PMA-stimulated neutrophils was activated by coreleased HOCl and most of the activity was destroyed after 1 h of stimulation. These results indicate that the HOCl/enzyme ratio must fall within a narrow range for activation to occur. In contrast to collagenase, purified progelatinase underwent negligible activation (2.5 +/- 1.2%) at HOCl/enzyme molar ratios less than 30 and was destroyed at higher ratios. Likewise no active gelatinase could be detected in supernatant from PMA-stimulated cells and almost all of the proenzyme was destroyed by HOCl after 60 min stimulation. Our results illustrate that only collagenase can be activated by HOCl in vitro and that gelatinase is much more sensitive to inactivation. Since a precise HOCl/enzyme ratio is required for collagenase activation it is doubtful whether effective enzyme regulation by HOCl could occur in vivo where various HOCl scavengers are present.     

The effects of antioxidants and hypohalous acid scavengers on neutrophil activation by hypochlorous acid-modified low-density lipoproteins

Hypochlorous acid-modified human blood low density lipoprotein (LDL–HOCl) was shown to stimulate neutrophils and to increase the luminol- (lm-CL) or lucigenin-enhanced chemiluminescence (lc-CL) of neutrophils. Antioxidants and HOCl scavengers (glutathione, taurine, cysteine, methionine, ceruloplasmin, and human serum albumin (HSA)) were tested for effects on lm-CL, lc-CL, H₂O₂ production, and degranulation of azurophilic granules of neutrophils. All agents used in increasing concentrations were found to decrease lm-CL produced by neutrophils upon stimulation with LDL–HOCl or subsequent treatment with the activator phorbol 12-myristate 13-acetate (PMA). The agents exerted a far lower, if any, effect on lc-CL and the H₂O₂ production by neutrophils in the same conditions. In the majority of cases, a decline in neutrophil chemiluminescence in the presence of the agents was not related to their effect on neutrophil degranulation, but was most likely due to their direct interactions with reactive halogen (RHS) or oxygen (ROS) species generated upon neutrophil activation or to myeloperoxidase (MPO) inhibition. Antioxidants and HOCl scavengers present in the human body were assumed to decelerate the development of oxidative or halogenative stress and thereby prevent neutrophil activation.     

Relative reactivities of N-chloramines and hypochlorous acid with human plasma constituents

Hypochlorous acid (HOCl), the major strong oxidant produced by the phagocyte enzyme myeloperoxidase, reacts readily with free amino groups to form N-chloramines. Since different N-chloramines have different stabilities and reactivities depending on their structures, we investigated the relative reactivities of three model N-chloramines and HOCl with human plasma constituents. TheN-chloramines studied were N(alpha)-acetyl-lysine chloramine (LysCA, a model of protein-associated N-chloramines), taurine chloramine (TaurCA, the primary N-chloramine produced by activated neutrophils), and monochloramine (MonoCA, a lipophilic N-chloramine). Addition of these chlorine species (100--1000 microM each) to plasma resulted in rapid loss of thiols, with the extent of thiol oxidation decreasing in the order TaurCA = LysCA > MonoCA = HOCl. The single reduced thiol of albumin was the major target. Loss of plasma ascorbate also occurred, with the extent decreasing in the order HOCl > LysCA > TaurCA > MonoCA. Experiments comparing equimolar albumin thiols and ascorbate showed that while HOCl caused equivalent loss of thiols and ascorbate, theN-chloramines reacted preferentially with thiols. The chlorine species also inactivated alpha(1)-antiproteinase, implicating oxidation of methionine residues, and ascorbate provided variable protection depending on the chlorine species involved. Together, our data indicate that in biological fluids N-chloramines react more readily with protein thiols than with methionine residues or ascorbate, and thus may cause biologically relevant, selective loss of thiol groups.     

Biological effects of Trichoderma harzianum peptaibols on mammalian cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Isolation of Salmonella resistant to hypochlorous acid from a poultry abattoir

Salmonella is a Gram-negative bacterium that has been implicated in several food-borne poisoning outbreaks. Poultry products are considered to be the major vehicles of food-borne poisoning caused by Salmonella . The resistance of Salmonella , isolated from different stages in a local poultry abattoir, to hypochlorous acid (HOCl) was studied. Isolates cultured in quarter strength Tryptone Soya Broth were treated with 72 ppm HOCl, incubated at 30 °C with shaking, and absorbance was measured at 660 nm every 20 min. A number of resistant isolates, which carried on growing following HOCl addition, were obtained. All the isolates from the bleeding stage were sensitive to HOCl, whereas those from the scalding stage were resistant to HOCl. Other stages had a population of resistant and sensitive isolates. It is evident that some Salmonella will grow in the presence of concentrations of HOCl deemed to be inhibitory. Hence, an effective concentration of HOCl must be applied in poultry abattoirs to ensure that all the Salmonella are eradicated.     

Flow effects on the viability and lysis of suspended mammalian cells

A mouse myeloma cell line growing in suspension was subjected intermittently to flow through a sudden contraction and turbulent flow in a capillary tube. The probability of lysis per pass through the capillary tube increased with average wall shear stress level and with residence time per pass in the tube. Lysis was first observed at a threshold average wall shear stress level of 1800 dyn/cm². Although the flow caused lysis, it had no effect on cell viability.