Enzymatic decolorization of melanin by lignin peroxidase from Phanerochaete chrysosporium.

Skin darkening results as a consequence of the accumulation of skin pigment melanin. To combat this, the amplitude of skin lightening agents are commercially available, most of which inhibit melanin synthesis. Decolorization of melanin is an alternative method of skin lightening. In this study, we show that lignin peroxidase (LiP), an extracellular enzyme purified from Phanerochaete chrysosporium NK-1 isolated from a forest soil can effectively degrade and decolorize melanin in vitro. Decolorization conditions including pH, temperature, incubation time, enzyme concentration, and mediator addition were investigated to optimize the reaction conditions. The results indicate that pH 3, 40 °C, 15 IU/ml, and 10 h incubation were the optimal conditions for the decolorization of the melanin. The use of the mediator, veratryl alcohol was also found effective to enhance the efficacy of the melanin decolonization, with up to 92% decolorization. The scanning electron microscopy results showed void spaces on the treated melanin granules as compared to the untreated sample, indicating the degradation of melanin. Changes in the fingerprint region of the melanin were observed. Between wavenumbers 1500-500 cm-1, for example, the presence of new peaks in the treated melanin at 1513, 1464, and 1139 cm-1 CH2, CH3 bend and C-O-C stretch represented structural changes. A new peak at 2144 cm-1 (alkynyl C≡C stretch) was also detected in the decolorized melanin. The cytotoxicity study has shown that the treated melanin and LiP have low cytotoxic effects; however, the mediator of veratryl alcohol could result in high mortality which suggests that its use should be meticulously tested in formulating health and skincare products. The findings of the study suggest that LiP produced by Phanerochaete chrysosporium has the potential to be used in the medical and cosmetic industries, particularly for the development of biobased cosmetic whitening agents.

Immobilized lignin peroxidase from Ganoderma lucidum IBL-05 with improved dye decolorization and cytotoxicity reduction properties.

Use of free microbial enzymes for bioremediation and other industrial applications has several disadvantages like low stability and non-reusability in repeated batch operations. Immobilized enzymes are stable, recoverable and reusable in industrial processes. In this scenario G. lucidum IBL-05 LiP was entrapped in Ca-alginate beads using optimum concentrations of Na-alginate (4%), calcium chloride (0.2M) and glutraldehyde (0.02%). Optimum pH (pH 5) and temperature (55°C) for entrapped LiP were improved as compared to free LiP. Catalytic behavior of LiP also significantly enhanced, as Km value (0.25mM) decreased and Vmax value (868.6µmol/min) increased after ca-alginate entrapment of LiP. Decolorization efficiencies of Sandal reactive dyes after treating with immobilized LiP were in the range of 80-93%. A significant reduction was observed in water quality parameters including, BOD (66.44-98.22%), COD (81.34-98.82%) and TOC (80.21-97.77%) values. The cytotoxicity values for heamolytic and brine shrimp lethality of dye solutions treated with Ca-alginate immobilized LiP reduced up to 2.10-5.06% and 5.43-9.23%, respectively. On the basis of reduced toxicity and cytotoxicity values, it was concluded that Ca-alginate beads entrapped LiP may be an effective biocatalyst for bioremediation of dye based textile industry effluents.

Characteristic features and dye degrading capability of agar-agar gel immobilized manganese peroxidase.

Immobilization of enzymes has been regarded as an efficient approach to develop biocatalyst with improved activity and stability characteristics under reaction conditions. In the present study, purified manganese peroxidase (MnP) from Ganoderma lucidum IBL-05 was immobilized in agar-agar support using entrapment technique. Maximum immobilization yield was accomplished at 4.0% agar-agar gel. The immobilized MnP exhibited better resistance to changes in pH and temperature than the free enzyme, with optimal conditions being pH 6.0 and 50 °C. The kinetic parameters Km and Kcat/Km for free and entrapped MnP were calculated to be 65.6 mM and 6.99 M(-1) s(-1), and 82 mM and 8.15 M(-1) s(-1), respectively. Thermo-stability was significantly improved after immobilization. After 120 h, the insolubilized MnP retained its activity up to 71.9% and 60.3% at 30 °C and 40 °C, respectively. It showed activity until 10th cycle and retained 74.3% residual activity after 3th cycle. The effects of H2O2, ionic strength and potential inhibitors on activity of free and immobilized enzyme were investigated. Moreover, the decolorization of three structurally different dyes was monitored in order to assess the degrading capability of the entrapped MnP. The decolorization efficiencies for all the tested dyes were 78.6-84.7% after 12h. The studies concluded that the toxicity of dyes aqueous solutions was significantly reduced after treatment. The remarkable catalytic, thermo-stability and re-cycling features of the agar-agar immobilized MnP display a high potential for biotechnological applications.

The cytotoxic activities of human hemoglobin and diaspirin crosslinked hemoglobin.

It is well known that hemoglobin (Hb) possesses many oxidative enzyme activities, including a pseudo-peroxidase activity. It has also been shown by many investigators that various peroxidases in the presence of hydrogen peroxide and a halide ion exert a potent cytotoxic activity toward various mammalian cell types. It has further been observed by various investigators that the administration of relatively large amounts of purified Hb or a Hb derivative to a host animal during resuscitation experiments leads to a number of unrelated types of tissue damage and cell damage in the host. The first objective of this investigation was to determine if the observed tissue and cell damage may be due to a cytotoxic activity that Hb may exert in vivo analogous to that of the peroxidases. We also showed some time ago that peroxidases are able to activate peritoneal macrophages to the cytocidal state. Hence, we also addressed the question whether or not Hb is able to activate macrophages in a similar manner. Our results were negative with regard to both questions. Further investigations indicated that, unlike the peroxidases, ferryl-Hb is unable to oxidize iodide to iodine at a measurable rate, which appears to be the reason for the lack of cytotoxic activity.

Induced hydrocephalus in postnatal rats following an intracerebral injection of ricin.

A single injection of peroxidase-labelled Ricinus communis agglutinin (RCA-HRP) was given intracerebrally in 5-day old postnatal rats to determine its effects on neural tissues. The rats were sacrificed at various time intervals ranging from 1 hour to 8 weeks after the injection. 5 days after the injection, the lateral ventricle ipsilateral to the injection was progressively enlarged. The size of the ventricle continued to expand so that 10-15 days after the injection the ventricle on the contralateral side was also affected. In longer surviving rats, i.e 3-8 weeks after the injection, both the ventricles were extremely dilated resulting in the thinning of the cerebral cortex. Scanning electron microscopy of the dilated ventricles showed signs of disruption of the ependyma in some regions. A number of cells including macrophages, neurons, glioblasts, astrocytes and oligodendrocytes were present on the ependyma. Their identification was confirmed by scanning- and transmission electron microscopy. Transmission electron microscopy of the cerebral cortex subjacent to the dilated ventricles showed the presence of many degenerating neurons, 2-5 hours after the injection of RCA-HRP. The neurons displayed typical features of degeneration, i.e. displacement of nucleus, dilatation of cisternae of rough endoplasmic reticulum, and swelling and disintegration of mitochondria. In conclusion, following a single intracerebral injection of RCA-HRP, drastic neuronal degeneration was elicited near the site of injection and this resulted in the dilatation of the lateral ventricles similar to hydrocephalus.

Antioxidation activity of tetrahydrobiopterin in pheochromocytoma PC 12 cells.

Rat pheochromocytoma PC 12 cells are susceptible to the oxidative toxicity caused by H2O2, nitrofurantoin, dopamine, and xanthine/xanthine oxidase reaction. The cytotoxicities of these agents are greatly reduced by the simultaneous presence of 0.1 mM tetrahydrobiopterin (BH4), 3 units/ml horseradish peroxidase, 0.2 mM NADH, and 0.1 units/ml sheep liver dihydropteridine reductase (DHPR). Individually, BH4, NADH and DHPR have no protection against H2O2 toxicity in PC 12 cells. Peroxidase alone offers 58% of protection if cells are incubated in the medium but only 3% in Dulbecco's phosphate buffered saline. The efficiency of the BH4-mediated antioxidation system in PC 12 cells is equal to or better than ascorbic acid and catalase, depending on the source of the reactive O2 species (ROS). The reactions responsible for the BH4-antioxidation system may consist of the non-enzymatic and the peroxidase-catalyzed reduction of H2O2 to H2O by BH4 and the regeneration of BH4 by DHPR using NADH as the cofactor. The components of this defence mechanism against ROS are all normal cellular constituents and are ubiquitous in nature. This DHPR-catalyzed redox cycling of BH4 may constitute an as yet little-known antioxidation system in mammalian cells.

In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins.

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.

Injurious effect of the eosinophil peroxide-hydrogen peroxide-halide system and major basic protein on human nasal epithelium in vitro.

Tissue injury is observed in allergic and nonallergic eosinophilic rhinitis, but the mechanism of this injury is unclear. Because eosinophils are prominent in biopsy specimens in these conditions, we hypothesized that they may participate in the injury process. Initially, we developed techniques to isolate and purify human nasal epithelial cells from turbinate biopsies to use as target cells for eosinophil granule products. Primary cultures from explants were characterized by electron microscopy and indirect immunofluorescence with a panel of primary monoclonal and polyclonal antibodies. These studies revealed the homogeneity of the cells and confirmed their epithelial nature. Cultured nasal epithelial cells were then exposed to either purified human eosinophil peroxidase, bromide, and glucose plus glucose oxidase, as a continuous source of hydrogen peroxide, or eosinophil major basic protein. Neither eosinophil peroxidase alone nor glucose plus glucose oxidase in the absence of eosinophil peroxidase were injurious, but the combined addition of eosinophil peroxidase, glucose/glucose oxidase, and bromide produced marked target cell lysis. This effect was time- and eosinophil peroxidase dose-dependent. Catalase and azide significantly inhibited the lysis of these cells, suggesting the eosinophil peroxidase-catalyzed products of halide oxidation mediated this form of injury. The addition of purified human eosinophil major basic protein also caused dose- and time-dependent lysis of the nasal epithelial cells but required longer incubation periods to effect injury. We hypothesize that the eosinophil peroxidase-hydrogen peroxide-halide system and major basic protein may injure the nasal epithelium in inflammatory conditions such as allergic and nonallergic eosinophilic rhinitis.

Comparative toxicity of the horse eosinophil peroxidase-H2O2-halide system and granule basic proteins.

Stimulated eosinophils release cytotoxic granule constituents, including eosinophil peroxidase (EPO) and a group of granule basic proteins (GBP). EPO reacts with H2O2 formed by the respiratory burst and a halide to form cytotoxic oxidants. The relative potency of the EPO-H2O2-halide system and the GBP is considered here. Horse eosinophils were induced to degranulate, the degranulation products were separated by chromatography on Sephadex G-50 and comparable volumes of the column fractions were tested for toxicity to Escherichia coli and the schistosomula of Schistosoma mansoni in the presence and absence of H2O2 and halides. Both the EPO system and GBP were toxic. However, the peak EPO fraction could be diluted 1000-fold at pH 7.0 and 5000-fold at pH 5.0, and with a 10-fold dilution at pH 7.0 incubation time could be reduced to 5 s, with retention of bactericidal activity in the presence of H2O2 and halides, whereas the peak GBP fractions diluted 10-fold had a small bactericidal effect at 1 h which increased with prolongation of incubation to 24 h. A less than 1 log fall in E. coli viable cell count was produced by the GBP fractions under all conditions as compared to total destruction (greater than 5 log fall) with the EPO system. A 1000-fold dilution of the peak EPO fraction was schistosomulocidal in the presence of H2O2 and halides, with toxicity observed at 2 h with a 10-fold dilution. In contrast, no schistosomulocidal activity was observed at 18 h with a 10-fold dilution of the GBP fractions. However, toxicity was observed with a 5- or 50-fold increase in GBP concentration with maximum toxicity observed with fractions between the two major protein peaks. Thus, under the conditions employed, the EPO-H2O2-halide system contributed to a considerably greater degree to the toxic activity of the granule components than did the GBP.

Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides.

Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system. The extent of such damage depends in part on the antioxidant defenses of the target cell. To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system. The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid. Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density. Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr. Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase. Reagent H2O2 could be replaced by glucose plus glucose oxidase. Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min. The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions. Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0. In the absence of halides, no lysis was observed. Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride. Fluoride was inactive. Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride. Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury. These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system. They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells. By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.

Oxidant membrane injury by the neutrophil myeloperoxidase system. II. Injury by stimulated neutrophils and protection by lipid-soluble antioxidants.

The stimulated human neutrophil can damage a variety of target cells, and in some models, a mechanism involving secretion of myeloperoxidase and H2O2 has been demonstrated. We explored the characteristics of this cell-cell interaction by using neutrophils and our recently described liposome model target cell system. Exposure of 51Cr-labeled liposomes to phorbol myristate acetate-stimulated human neutrophils resulted in release of 25 to 30% of the radioactivity. 51Cr release was abrogated by omission of the neutrophils, the phorbol ester or halide (iodide), replacement of the phorbol by an inactive congener, or addition of azide, cyanide, or catalase. Neutrophils from patients with hereditary absence of myeloperoxidase (MPO) or a failure of H2O2 formation (chronic granulomatous disease) did not cause liposome lysis unless purified MPO or a source of H2O2, respectively, was added. These data indicate that 51Cr release from liposomes is a consequence of the secretion of MPO and H2O2, which combine with extracellular halides to form a membrane lytic system. The influence of liposome composition on injury was then examined, with a focus on physiologically relevant lipid soluble antioxidants. Liposomes containing either alpha-tocopherol (0.33 to 1.67% of molar fraction of lipid) or beta-carotene (1.67% of molar fraction of lipid) were markedly resistant to lysis by the cellfree MPO-H2O2-chloride system. When the major structural lipid phosphatidyl choline was replaced by dipalmitoyl phosphatidyl choline, a synthetic phospholipid with no oxidizable double bonds, the resultant liposomes were totally resistant to lysis by the MPO-H2O2-chloride system. The addition of iodide to this system (i.e., both chloride and iodide present) changed the pattern of protection dramatically in that alpha-tocopherol and beta-carotene were no longer protective and the resistance of dipalmitoyl phosphatidyl choline liposomes was partial rather than complete. In contrast to iodide, the addition of bromide or thiocyanate did not have a major effect on the protection by antioxidants. Finally, we demonstrated protection by alpha-tocopherol or dipalmitoyl phosphatidyl choline against liposome lysis by phorbol-activated neutrophils. These studies illustrate the use of model phospholipid membranes in the characterization of oxygen-dependent cell-mediated cytotoxicity. Activated neutrophils lyse liposome targets through a MPO-dependent mechanism. Target properties, especially the content of lipid-soluble antioxidants, have a marked influence on susceptibility to lysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific antibody-forming cells in bronchus-associated lymphoid tissue (BALT) and lung of the rat after intratracheal challenge with horseradish peroxidase.

After intratracheal or subcutaneous priming with horseradish peroxidase (HRP), no anti-HRP-forming cells were present in the bronchus-associated lymphoid tissue (BALT) or the lung of the rat. After intratracheal priming and intratracheal boosting with HRP no specific antibody-forming cells were observed either in the BALT or the lung. A few blast cells containing anti-HRP antibody were found in paratracheal lymph nodes, which is possibly the source of anti-HRP-forming cells. After subcutaneous priming in the hind footpad and intratracheal boosting specific antibody-forming cells were present in both the BALT and in the lung. In the BALT these cells were found peripherally, and in the lung perivascularly and peribronchiolarly. The simultaneous appearance of these anti-HRP-forming cells at both sites, their localization and their morphology strongly indicate that they are recruited from the circulation and not formed in situ; the probable source is the popliteal lymph node.

Toxic effect of the peroxidase-hydrogen peroxide-halide antimicrobial system on Mycobacterium leprae.

Mycobacterium leprae are killed by myeloperoxidase (or eosinophil peroxidase), H2O2, and a halide, thus suggesting a mechanism for their destruction by peroxidase-containing phagocytes.

Horseradish peroxidase acute ototoxicity and the uptake and movement of the peroxidase in the auditory system of the guinea pig.

When guinea pig cochlea was perfused in vivo with a solution of 1% horseradish peroxidase (HRP) in artificial perilymph, the enzyme was found in the basilar membrane, spiral limbus, some outer and inner hair cells and some supporting cells and it was gradually cleared away with time. Acute signs of cell damage included swelling, vacuolization and diffuse labeling of some hair cells, but stereocilia remained normal in configuration. Albino melanocytes of the spiral ligament were also damaged, and vacuolization of Reissner's membrane occurred after 10% HRP. Both concentrations caused a gradual decline in CM, showing that HRP is acutely ototoxic but its mode of action is unknown. No retrograde transport of HRP to spiral ganglion cells or to brain stem neurons occurred, but some brain stem neurons took up HRP from the neuropil following diffusion from the cochlea.

Peroxidase-H2O2-halide system: Cytotoxic effect on mammalian tumor cells.

Myeloperoxidase, H2O2, and a halide constitute a potent antimicrobial system. A cytotoxic effect of this system on a line of mouse ascitic lymphoma cells (LSTRA) is demonstrated here using four different assay systems: 51Cr release, trypan blue exclusion, inhibition of glucose C-1 oxidation, and loss of oncogenicity for mice. Deletion of each component of the system, preheating the peroxidase, or addition of azide, cyanide, or catalase abolished the cytotoxicity. Myeloperoxidase was effective with either chloride or iodide as the halide, while lastoperoxidase was effective with iodide but not chloride. The iodinated thyroid hormones, triiodothyronine and thyroxine, could substitute for the halide, and H2O2 could be replaced by a peroxide-generating enzyme system such as glucose and glucose oxidase or by H2O2 producing bacteria such as pneumococci or streptococci. The possibility is raised that the peroxidases of inflammatory cells and certain biologic fluids may affect tumor initiation or growth in vivo.