oxidative metabolism canova

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Canova, a Brazilian medical formulation, alters

oxidative metabolism of mice macrophages

Carolina C. de Oliveira, Simone M. de Oliveira, Lyris M.F. Godoy,Juarez Gabardo, Dorly de F. Buchi*

Laboratorio de Estudos de Celulas Inflamatorias e Neoplasicas, Departamento de Biologia Celular,

SCB, Universidade Federal do Parana; Curitiba, PR, Brasil

Accepted 18 August 2005Available online 4 January 2006

KEYWORDSReactive oxygenspecies;Nitric oxide;Macrophages;Canova

Summary Macrophages play a significant role in the host defence mechanism.When activated they can produce reactive oxygen species (ROS) as well as relatedreactive nitrogen species (RNS). ROS are produced via NAD(P)H oxidase whichcatalyzes superoxide (OK2 ) formation. It is subsequently converted to hydrogenperoxide (H2O2) by either spontaneous or enzyme-mediated dismutation. Nitric oxidesynthase (NOS) catalyzes nitric oxide (NO) formation. Canova (CA) is a Brazilianmedication produced with homeopathic techniques, composed of Aconitum, Thuya,Bryonia, Arsenicum, Lachesis in distilled water containing less than 1% ethanol.Previous studies demonstrated that CA is neither toxic nor mutagenic and activatesmacrophages decreasing the tumor necrosis factor-a (TNFa) production. In this assaywe showed that macrophages triggered with Canova increased NAD(P)H oxidaseactivity as well as that of iNOS, consequently producing ROS and NO respectively.Cytochrome oxidase and peroxisomes activities were inhibited by NO. As NO and OK2are being produced at the same time, formation of peroxynitrite (ONOOK) may beoccurring. A potential explanation is provided on how treatment with Canova mayenhance immune functions which could be particularly important in the cytotoxicactions of macrophages. CA can be considered as a new adjuvant therapeuticapproach to known therapies.Q 2006 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Macrophages play a significant role in the hostdefence mechanism. When activated, they produceand release reactive oxygen species (ROS) inresponse to stimulation with various agents1 andcan inhibit the growth of a wide variety of tumorcells and micro-organisms.2 Another importantpathway stimulated involves the generation of

Journal of Infection (2006) 52, 420432

www.elsevierhealth.com/journals/jinf

0163-4453/$30.00 Q 2006 The British Infection Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.jinf.2005.08.017

* Corresponding author. Address: UFPR, Centro Politecnico,SCB, Dpto de Biologia Celular, sala 215, Jardim das Americas-Curitiba, PR-Brazil CEP 81531-980. Tel.: C55 41 361 1770; fax:C55 41 361 1568.

E-mail addresses: [email protected] (C.C. de Oliveira),[email protected] (D.F. Buchi).
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nitric oxide (NO) and related reactive nitrogenspecies (RNS).3

Oxygen metabolites play an important role in theintracellular and extracellular killing of micro-organisms, and may also serve as mediators of theimmune system and modulators of cellular activi-ties such as adhesion, phagocytosis and signal

transduction.4

In this metabolic pathway, oxygenis enzymatically reduced to superoxide anion (OK2 )through a series of one-electron reductive reactionscatalyzed by the NAD(P)H oxidase system.5,3 OK2 issubsequently converted to hydrogen peroxide(H2O2) by either spontaneous or enzyme-mediateddismutation via superoxide dismutase (SOD).NAD(P)H is recognized as one of the primaryenzyme complexes involved in the anti-pathogenaction of phagocytic cells. It is a membraneassociated multisubunit enzyme.6 In unstimulatedcells, components of NAD(P)H oxidase are distrib-uted throughout the membrane and the cytosol.7

NO is a diatomic mediator generated from thefive-electron oxidation of L-arginine and molecularoxygen catalyzed by the nitric oxide synthase (NOS)family of enzymes.8 Two of these are constitutivelyexpressed in vascular endothelial cells (eNOS ortype III NOS) and in neurones (nNOS or type I NOS),whereas the expression of a third isoenzyme (iNOSor type II NOS) is inducible in a variety of cells. NOproduction has also been detected in mitochondrialpreparations and the existence of a mitochondrialNOS (mtNOS) associated with the inner mitochon-drial membrane, has been postulated.9 The con-

stitutive forms are low-activity enzymes thatgenerate small amounts of NO as signaling mol-ecules. Once induced, iNOS produces large amountsof NO which accounts for its microbicidal andtumoricidal activities.10 Unlike active NAD(P)Hoxidase, NO syntase is soluble.9

A medical formulation called Canova (CA) is aBrazilian medication based on homeopathic tech-niques. It has a patented formulation sold underprescription in specialized drugstores. CA stimu-lates the host defence favoring an immunologicresponse against several pathologic states throughactivation of the immune system. Previous studiesdemonstrated that CA activates macrophages bothin vivo and in vitro. In addition, Tumor NecrosisFactor-a (TNFa) production is significantly dimin-ished.11 CA modulatory effects were observed inexperimental infection both in vivo and in vitro byLeishmania amazonensis controlling infection pro-gression and limiting it dissemination.12 Sarcoma180-bearing mice treated with CA showed reductionin sarcoma size and a significant infiltration oflymphoid cells, granulation tissue and fibrosissurrounding the tumor. Besides T CD4, T CD8, B

and NK cells increased both in normal-treated andS180-treated mice groups.13 Clinical reports shownsuccessful results when CA is employed in diseaseswhere the immune system is depressed due to itsmodulatory capacity.14 Moreover, it is neither toxicnor mutagenic.15 CA is indicated in deseases werethe immune system is depressed such as acquired

immunodeficiency syndrome (AIDS), hepatitis andneoplasia. Thus, providing an insite into theusefullness of this medicine may contribute toknowledge of how the immune system works.

The role of ROS/RNS on bacterial killing andtissue damage has long been explored.1 It followsthat mechanisms involving ROS/RNS productionmay have important implications in new thera-peutic approaches. We now evaluate whether theinfluence of CA on mouse macrophages isaccompanied by alterations linked to oxygen andnitrogen metabolism.

Materials and methods

Canova (CA)

Canova is a commercial medicine that represents anew form of immunomodulatory therapy andfollows Hahnemanns ancient homeopathic tech-niques. Canova is an aqueous, colourless andodorless solution produced and sold in Brazilianauthorized drugstores.

Mother tinctures are purchased from authorizedagencies indicated by the Brazilian Health Ministry.These agencies assure the quality (endotoxin free)and physico-chemical composition of its products.Starting from the original mother tincture (in thecase of a plant this is an ethanolic extract) severaldinamizationssucussion (shaking) and dilution indistilled waterare performed. Decimal dilutions(dH) are prepared. The final commercial product,Canova, is composed of 11 dH Aconitum napellus(Ranunculaceae), 19 dH Thuya occidentalis (Cupre-saceae), 18 dH Bryonia alba (Curcubitaceae), 19 dH

Arsenicum album (arsenic trioxide), 18 dH Lachesis

muta (Viperidae) and less than 1% ethanol indistilled water (www.Canovadobrasil.com.br).

In our experiments we used the commercialproduct purchased from Canova do Brasil.

Animals

Six8 week old male Swiss mice from the CentralAnimal House of the Universidade Federal doParana received a standard laboratory diet (Pur-inaw) and water ad libitum. All recommendations of

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the National Law (N8 6.638, November, 5th 1979)for scientific management of animals wererespected. Experiments were carried out at theLaboratorio de Pesquisa em Celulas Neoplasicas eInflamatorias, UFPR, which has a managementprogram for produced residues.

CA treatment

All experiments were performed at least threetimes in quadruplicate and with two control groups.These were mice without any treatment and micetreated with an ethanolic solution (less than 1%).The treatment groups were held according to aprevious description.11

In vivo treatment:Mice were treated at daily intervals for 7 days.Seven ml/g of commercial CA were injected

subcutaneously.

In vitro treatment:Three-h cultured cells were treated with CA(200 ml/ml), and after 24 h a new dose of 20 ml/mlwas given without replacing the medium. Treat-ment was carried out for 48 h in vitro.

Canova and the ethanolic solution were vigor-ously shaken, a process named sucussion, immedi-ately before treatment.

Cell culture preparation

Macrophages were washed from peritoneal cavitieswith 10 ml of either cold phosphate buffer solution(PBS) or Hanks buffer salt solution (HBSS), pH 7.4.Harvested peritoneal cells were counted using aNeubauer chamber. The macrophages were incu-bated at 37 8C under 5% CO2 for 15 min and the non-adherent cells were removed by washing with PBS.Dulbeccos Modified Eagles Medium (DMEM) sup-plemented with 10% fetal bovine serum (FBS),50 mg/ml penicillin and 100 U/ml gentamicin wasadded to culture and handled according to theprocedures required by each experiment. Accord-

ingly,O90% of adherent cells were macrophages11

and the preparation was not further purified.

Immunogold to iNOS detection

Cells (2!107 cells/group) of in vivo and in vitrotreatment were fixed for 1h in freshly prepared0.05% glutaraldehyde, 2% paraformaldehyde/PBS,and then rinsed and incubated for 30 min in 50 nMammoniun chloride/PBS. Monoclonal antibodyagainst mouse iNOS (Sigma) was diluted (1:100) in

0.01% Triton-X/ PBS where cells were incubated for1 h at 25 8C. Before adding the secondary antibody,cells were rinsed five times with PBS. Incubationwith Protein Agold 10 nm (Sigma) was performedfor 30 min at room temperature, in 0.01% Triton-X/PBS. Cells were re-fixed in 2.5% glutaraldehyde, 4%paraformaldehyde/PBS for 30 min and processed to

electron microscopy. Cells were post fixed in 1%OsO4, dehydrated in acetone andembedded in Epon.Ultrathin sections were stained with uranyl acetateand lead citrate and observed in a Jeol 1200 EXIItransmission electron microscope at the Centro deMicroscopia Eletronica, UFPR. A GATAN imageanalyser was used to produce theelectronmicrographs.

Ultrastructural cytochemistry to detectNAD(P)H oxidase activity

Macrophages treated in vivo and in vitro with CAwere processed for detection of NAD(P)H oxidaseactivity (2!107 cells/group). This technique isbased on the reaction of H2O2 generated by cellswith cerium chloride, which results in a precipitateof cerium perhydroxide (Ce-[OH]2OOH).

16 Briefly,adherent cells were washed with 0.1 M TRIS-maleate buffer (pH 7.5) containing 7% sucrose, at4 8C. After washing, the cells were incubated for10 min at 37 8C with the same buffer containing1 mM 3-amino-1,2,4 triazolecatalase inhibitor(AT), and subsequently incubated with a newsolution for 20 min at 37 8C in 0.1 M TRIS-maleatebuffer (pH 7.5) supplemented with 7% sucrose,0.71 mM NADH as the substrate, 2 mM CeCl3, asthe capture agent, and 10 mM AT. The solution usedas enzyme control lacked the enzyme substrate. Theenzyme control group is used to gain some insightinto the specificity of the reaction. After incu-bation, the cells were washed twice with buffer andfixed in a solution containing 1% glutaraldehyde, 4%paraformaldehyde and 5 mM CaCl2 in 0.1 M cacody-late buffer, pH 7.2. The cells were gently scraped offwith a rubber policeman and processed to trans-mission electron microscopy as described above.

The cells were observed without uranyl acetate andlead citrate stain to guarantee the results.

Ultrastructural cytochemistry to detectcytochrome oxidase and catalase

The peroxisomal enzyme marker catalase and thecytochrome oxidase activity were detected basedupon the oxidative polymerization of 3,3 0-diamino-benzydine (DAB) to an osmiophilic reaction product(DAB precipitation).1719 Reaction products

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indicative of catalase and cytochrome oxidaseactivity were observed in both in vivo and in vitroassays. Cells (2!107 cells/group) were rinsed at4 8C with 0.1 M cacodylate buffer (pH 7.5) contain-ing 5% sucrose and fixed for 1 h at 25 8C in a solutionwith 1% glutaraldehyde in 0.1 M cacodylate buffer(pH 7.2) containing 5% sucrose. The cells were then

rinsed twice with 0.1 M cacodylate buffer (pH 7.2)containing 5% sucrose, and washed twice more with0.05 M TRIS-HCl buffer (pH 7.6) containing 5%sucrose. The reaction medium containing 5 mg3,3 0-diaminobenzidine (DAB) in 0.05 M TRIS-HClbuffer (pH 7.6) were added and the cells wereincubated for 1 h at 37 8C and subsequently incu-bated for 10 min with 0.05 M TRIS-HCl buffer (pH7.6). The cells were gently scraped off with a rubberpoliceman and processed according transmissionelectron microscopy protocol as described above.These cells were also observed without stain.

Micro-organisms

Trypanosoma cruzi Dm28c20 clones epimastigoteswere maintained and grown in liver infusiontryptose (LIT) medium at 28 8C. To obtain meta-cyclic tripomastigotes, the epimastigotes in thelate exponential growth phase were harvested fromLIT medium by centrifugation and incubated for 2 hin triatomine artificial urine (TAU) (190 mM NaCl,17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.035%NaHCO3, 8 mM sodium phosphate pH 6.0). Then,parasites were transferred to cell culture bottles

containing TAU3AAG (TAU supplemented with50 mM L-glutamate, 10 mM L-proline, 2 mM L-aspartete and 10 mM D-glucose) and incubated for34 days at 28 8C.21 L. amazonensis (MHOM/BR/73/M2269)22 promastigotes were maintained in Tobieand Evans biphasic medium at 24 8C. Parasites werecultivated in RPMI 1640 medium supplemented with20% FBS for 7 days to provide multiplication andused at log phase.23

Macrophage-micro-organisms interaction

Macrophages were cultivated for 24 h and thenallowed to interact with the intracellular parasitesat a ratio of 10:1 for 2 h in medium without FBS. Theparasites were washed out and the macrophageswere cultivated with standard medium for 24 or72 h to detect NO production.

Nitric oxide (NO) production

The NO generation was estimated by samplingculture supernatants for nitrite, which is a stable

product of NO reaction, as described elsewhere.24

Macrophages treated in vivo and in vitro (5!105 cells/well) were plated into 96-well tissueculture plates. After 48 h, aliquots of 100 ml ofcell-free supernatant were mixed with an equalvolume of Griess-reagent (0.5% sulfanilamide and0.05% N-1-naphtyl ethylenediamine dihydrochlor-

ide in 2.5% phosphoric acid) in 96-well tissue cultureplates and incubated for 10 min at 25 8C. 50 ng/mlLPS and 26 U/ml IFN-g were added as a positivecontrol for NO production. Optic density of thesamples was subsequently measured at 550 nm at amicroplate reader (BIO-RAD). The nitrite concen-tration was determined by reference to a standardcurve using sodium nitrite (1080 mM) diluted inculture medium.

Superoxide anion (OK2) detection

Reduction of ferricytochrome c was used tomeasure rates of formation of OK2 in culturesupernatant as described elsewhere.25 Formeasurement of OK2 , adherent cells treated in vivoor in vitro (5!105 cells/well) were incubated inHBSS containing ferricytochrome c (80 mMSigma)in the presence or absence of 1 mg/ml phorbolmiristate acetate (PMA). Since PMA is able to induceOK2 production by macrophages

26 it was used aspositive control. Absorbance was measured at550 nm in a microplate reader (BIO-RAD) and theextinction molar coefficient 3Z2.1!104 MK1 CaK1

was used to determine reduced ferricytochromec. Results are expressed as nmol OK2 /10

6 cells.

Hydrogen peroxide (H2O2) measurement

Production of H2O2 by macrophages after in vivo orin vitro treatment was quantified based on thehorseradish peroxidase-dependent oxidation ofphenol red by H2O2.

5 Macrophages (3.5!105 cells/well) were incubated in horseradish peroxidase(15 U/ml, type IV-A-Sigma) and 194 mg/ml phenolred solution dissolved in HBSS at 4 8C, briefly beforethe start of the experiment. 1 mg/ml of PMA wasadded as a positive control of H2O2 production.

26

The plates were incubated at 37 8C for the desiredtime interval (60 and 90 min) and the reactionstopped by adding 10 ml 1 M NaOH aqueous solutionper well. The absorbance of cell-free culturesupernatant was read at 620 nm at a microplatereader (SLT Lab Instruments 340 ATC). The H2O2concentration was determined by reference to astandard curve using 150 nmol of H2O2 in a solutioncontaing 15 U/ml peroxidase, 194 mg/ml phenolred in HBSS.



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Statistical analysis

Results are expressed as the meansGstandarddeviation (SD). Data were submitted to analysis ofvariance (ANOVA) and Tukey test (P!0.05) todetermine the statistical significance of the inter-group comparisons. Data are representative of

three independent experiments.

Results

Results from in vivo and in vitro experiments werevery similar. Our results also demonstrated nostatistical differences between the control groupand the ethanolic aqueous solution group, thesethus being referred only as the control group.Significant differences were observed in the treatedgroup when compared to the control group. Cells

from the control groups were mainly residentmacrophages (Fig. 1(A)) and few activated macro-phages were also present, as already described.11

Almost all cells from the treated group wereactivated, as defined by morphological alterations(Fig. 1(B)).

Immunogold to iNOS detection

The signal molecule NO is synthesized on demand,after enzyme activation. The inducible NOS (iNOS)once expressed, produces NO for long periods (hoursto days).27 For a better understanding, the mech-

anism through Canova acts, namely the expressionof iNOS from peritoneal macrophages, was ana-lysed. There was a marked difference between the

morphology of control and treated cells. The controlcells were smaller, had few vesicles and membraneprojection compared with the treated cells. Macro-phages treated with Canova contained increasediNOS levels when compared with the control group.The enzyme was found on the cytoplasm locatedmainly near the nuclei (Fig. 2(A)), mitochondria

(Fig. 2(B)) and vesicles (Fig. 2(C) and (D)). Thecontrol group only contained iNOS in the fewactivated macrophages detected.

Ultrastructural cytochemistry to detectNAD(P)H oxidase activity

NAD(P)H oxidase is an electron transport chain thatuses NAD(P)H as a electron donor to reduce oxygen(O2) to superoxide (O

K2 ) and hydrogen peroxide

(H2O2).26 Reaction products (cerium precipitated)

indicative of NAD(P)H oxidase activity were

detected in both in vivo and in vitro assays. Whenthe enzyme substrate was eliminated from theincubation medium (enzyme control group), noreaction product was observed (Fig. 3(A)) in bothgroups. It is clear that the generation of thereaction product is strongly dependent on thepresence of exogenous NAD(P)H, there being aspecificity of this reaction. Occasional deposits ofreaction product were detected in the controlgroup. When found, they were located mainly inthe few activated cells inside vesicles and on theexternal surface of the plasma membrane(Fig. 3(B)). No other cytoplasmic organelles were

reactive for H2O2. The appearance of treated cellswas markedly different from the control cells.Deposits of reaction product were mainly found

Figure 1 Electronmicrographs showing morphological characteristics of peritoneal macrophages. (A) Representativecell from control group showing typical resident morphology. (B) Activated macrophage representative of treatedgroup. Among these cells approx. 85% had typical activated morphology. Data represent three independentexperiments.



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inside vesicles (Fig. 3(C) and (D)) and these werestronger than in the control. Equal amounts ofelectron-dense material was detected in theexternal surface of the plasma membrane fromtreated and control cells. Other cytoplasmicorganelles were not found to contain H2O2 usingthis method.

Ultrastructural cytochemistry to detectcytochrome oxidase and catalase

Cytochrome oxidase catalyses the transfer ofelectrons from cytochrome c to oxygen formingwater in the mitochondria. Catalase is involved inthe peroxidative reaction of H2O2 inside peroxi-somes. DAB donates electrons in both reactions.18,19

Reaction products (DAB precipitated) indicative ofcatalase and cytochrome oxidase activity weredetected in both in vivo and in vitro assays mainly

with the control cells. The reaction products foundin the control group were evident and distributedeither inside peroxisomes or inner mitochondrialmembranes (Fig. 4(A)(C)). Generally no positivereaction was found in the treated group. However,when found, it was weak as seen in Fig. 4(D).

Nitric oxide (NO) production

NO has been shown to be part of the oxidative warchest of the immune system by virtue of itsinvolvement in anti-tumor and anti-pathogen hostresponse.28 It is known to be secreted by macro-phages in response to IFN-g stimulation,29 and LPS isrecognized as a co-signal in the induction of NOS.30

NO production was evaluated on in vivo and in vitrotreatment with CA, in the presence or absence ofLPS and IFN-g. Both treatments resulted in amoderate but significant up-regulation of NO

Figure 2 Immunogold to iNOS detection on treated macrophages. Canova treated cells had increased iNOS levelswhen compared to control group. This enzyme was located mainly near the nucleus (A), mitochondria (B), and insidevesicles (C) and (D). Arrows pointing at gold 10 nm are representative of iNOS localization. Data represent threeindependent experiments.



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production by macrophages. The NO productionincreased after 48 h, when treated with CA. Ontriggering the cells with LPS and IFN-g, as expected,control cells increased their NO production, butmacrophages from the treated group significantlyincreased the NO production under this condition

(Fig. 5(A)).

Nitric oxide production in presence ofparasites

Nitric oxide is one of the main molecules that actagainst intracellular parasites. NO can act directlyon parasites, causing toxic and inhibitory effects onseveral cellular processes as growing and multipli-cation. The capacity to inhibiting NO production hasbeen demonstrated to several intracellular

parasites such as Leishmania spp.31, Toxoplasmagondii32 and fixed T. cruzi.33

NO production by macrophages of control groupdecreased in presence of parasites, suggesting aninhibitory process. However, this decrease was notobserved in macrophages from CA treated group.

Statistical tests showed that CA treatment led to asubstantial enhancement of NO production bymacrophages during interaction with L. amazonen-sis (46 and 32%), T. cruzi epimastigote (44 and 18%)and T. cruzi trypomastigote (32 and 5%) after 24 and72 h, respectively (Fig. 5(B) and (C)).

Superoxide anion (OK2) detection

Phagocytic cells respond to a variety of membranestimulants by the production and release of

Figure 3 Electronmicrographs showing ultrastructural cytochemical localization of macrophages NAD(P)H oxidaseactivity. These cells were observed without stain. (A) Absence of reaction product in macrophage from enzyme controlgroup confirming the specificity of this reaction. (B) Cell from control group showing occasional positive reactionproducts. Note many vesicles with no reaction products. (C) and (D) Treated cells having an intense positive reactionmainly inside vesicles. Arrows point the reaction product representative of NAD(P)H oxidase activity. Data representthree independent experiments.



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a number of reactive oxygen reduction products.This response is initiated by reduction of O2 to O

K2 .

34

OK2 detection is based on ferricytochrome creduction. The time course for OK2 extracellularrelease from control was compared to that oftreated cells. At first, both groups had the samerate of OK2 production. After 15 min, the release offrom treated cells diminished considerably OK2(Fig. 6(A)). Under PMA stimulation, a significant

decrease on OK2 production was observed comparedwith the first reading (Fig. 6(B)).

Hydrogen peroxide (H2O2) measurement

Reactive molecules, such as H2O2, are producedfrom macrophages after various stimuli and havewell-established roles in anti-microbial defence aswell as in cell signaling.30 Quantification of H2O2production is based on the horseradish peroxidase-dependent oxidation of phenol red which is assayed

by an increased absorbance at 620 nm5. Themeasurement of H2O2 on culture supernatant fromtreated and non-treated macrophages gave nostatistically significant differences in both treat-ments (data not shown).

Discussion

Macrophages play an essential role in host defenceagainst infection and tumoral cells. A large body ofdata indicates that macrophages must be activatedin order to achieve efficiency. Recent studies havedescribed the effects of Canova on macrophages.About 86% of treated macrophages were activatedwhen observed in light microscopy and transmissionelectron microscopy.11 The increased responsecapacity of activated macrophages is a result, inpart, of the increase capacity of these cells toproduce oxygen radicals; thus the oxidative

Figure 4 Ultrastructural cytochemical detection of catalase and cytochrome oxidase activity. These cells were

observed without stain. (A)(C) Showing DAB precipitated representative of catalase (peroxisomes) and cytochromeoxidase (mitochondria) activity in control cells. (D) Treated cells showing weak positive reaction products. Arrowspointing at positive reaction products. Data are representative of three independent experiments.



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metabolism of macrophages treated with Canovawas evaluated. Results are summarize in Fig. 7.

NO is formed biologically through the oxidationof L-arginine by nitric oxide synthases. It is acytotoxic product of activated macrophages,along with other reactive oxygen species thathave been shown to be involved in numerousregulatory functions.28,35 In vivo and in vitro assaysshowed that Canova acts on macrophages

up-regulating nitric oxide (NO) production. Theseeffects seem to be cumulative with LPS and IFN-g,since NO production increased significantly underthis condition (Fig. 5(A)). The in vitro adminis-tration of CA led to a substantial enhancement ofNO production by macrophages after interactionwith L. amazonensis and both forms of T. cruzi for24 and 72 h (Fig. 5(B) and (C)). It seems that CAnot only neutralizes the inhibitory effects ofparasites but also stimulates the oxidative metab-olism, as discussed above. The present study hasshown that the increase in NO production isaccompanied by an increase in iNOS detected.The enzyme was found on the cytoplasm locatedmainly near vesicles and mitochondria (Fig. 2(B)(D)). NO and reactive oxygen species (ROS) areproduce d, under a variet y o f biolog icalcon ditions and th ey are critical in h ostdefence not only because they can damagepathogens and tumoral cells but also since theyare immunoregulatory.30,36

Figure 5 NO production. Macrophages were obtainedeither from 7-day-treated mice with 7 ml/g of MC or fromnon-treated animals. Nitrite (NOK2 ) concentration (mM) insupernatants was determined with Griess reagent. (A)Control versus treated group in the presence or absenceof LPS and IFN-g after 48 h. (B) and (C) Parasites wereadded (10:1) after 24 h of culture and allowed to interactwith macrophages during 2 h. Nitrite (mM) concentrationin supernatants was determined 24 (B) and 72 (C) h afterinteraction. Results are expressed as meanGSD (signifi-cantly difference from the respective control group byTukey test *P!0.05). Data represent three independent

experiments.

Figure 6 Time course of OK2 extracellular release.Macrophages were obtained either from 7-day-treatedmice with 7 ml/g of MC or from non-treated animals. OK2concentration in supernatants was determined by ferri-cytochrome c reduction. Results are expressed as meanGSD (significantly different from the control group byTukey test *P!0.05). (A) Control and treated groups hadthe same rate of OK2 production. After 15 min, the releaseof OK2 from treated cells diminished considerably. (B)Under PMA stimulation a significant decrease on OK2

production was observed compared with the first readvalue. Data represent three independent experiments.



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The enhanced capacity of activated mouseperitoneal macrophages to secrete ROS may bedue to an increased affinity of their oxidase forNAD(P)H.37 Ultrastructural cytochemical detectionof NAD(P)H oxidase activity was performed andcharacterized by a local cerium precipitate. Ultra-thin sections of the material were observed withoutstain, so that electrondense markers are indicativeof a positive enzyme reaction. NAD(P)H oxidase is

normally dormant in resident macrophages, but canbe rapidly activated by a variety of stimuli. Whenthe phagocyte is activated, the cytosolic subunitsmigrate to the membranes, where they bind to themembrane-associated subunits to assemble theactive oxidase resulting in the delivery of itsproducts into vesicles and extracellular environ-ment.10 The control group showed electrondenseproducts in the few activated macrophages found(Fig. 3(B)). Canova, in some way, activates thispathway because in the treated cells we observed,mainly in vesicles, stronger positive reactionproducts indicative of NAD(P)H oxidase activity(Fig. 3(C) and (D)). No other cytoplasmic organelleswere found in both groups to contain hydrogenperoxide (H2O2) by this method. The externalsurface of the plasma membrane also presentedelectron-dense material, but this was apparentlyequally distributed in the groups. This was con-firmed by measuring H2O2 in the culture super-natant, which showed no statistical significantdifferences between the groups (data not shown).Quantification of H2O2 on culture supernatant wasbased on the horseradish peroxidase-dependent

oxidation of phenol red, which was assayed byincreased absorbance at 620 nm. The major sourceof H2O2 in cells arises from either spontaneousconversion of superoxide anion (OK2 ) or via theaction of the enzyme superoxide dismutase whichcatalyses the formation of H2O2 from O

K2 .

2,6 WhenOK2 is produced and released to the outside of thecell, the most reliable method for measurement ofOK2 is the reduction of ferricytochrome c.

1 Detection

of OK

2 in the culture supernatant showed nostatistical differences between control and treatedgroups (Fig. 6(A) and (B)). These results suggestthat Canova is stimulating intracellular productionof ROS, thus favoring a specifically immunologicalresponse. A state of moderately increased levels ofintracellular ROS is referred to as oxidative stress.38

ROS have received increasing recognition for theirrole in host defence,4 and as second messengers inthe signaling pathways of macrophages. This resultsin a broad array of physiological responses1 such asmodulation of anti-oxidant levels, induction of newgene expression and protein modification.38

Cytochrome oxidase activity was determined byultrastructural cytochemistry. We found enzymeinhibition in Canova treated cells (Fig. 4(D)).Cytochrome oxidase is an enzyme that catalyzesthe transfer of electrons from its reduced substrateto molecular oxygen to form water, playing acritical role in energy metabolism.39 It is knownthat NO reacts with a number of molecules, being asmall gaseous free radical that binds readily toheme iron. The enzyme cytochrome oxidase con-tains three redox-active metal sites including

Figure 7 Activated macrophage summarizing the effects of Canova. It increased NAD(P)H oxidase in vesicles thusincreasing H2O2 within then. Canova also increased iNOS expression increasing NO production. NO can react with O

K

2forming ONOOK besides inhibiting cytochrome oxidase. White arrow Z formation; grey arrow, probable formation;black arrow, inhibition.



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a heme iron that catalyzes electron transfer tooxygen reduction sites.40 NO can bind to cyto-chrome oxidase thereby controlling cellular func-tions.9 As we found an up-regulation of NOproduction, we can assume that NO is interactingwith this enzyme. Cells producing NO as amessenger, activator, or modulator are faced with

its potential toxicity. Free NO in the cell environ-ment for a long enough periods can ultimatelyinduce apoptosis and cell death.41 Therefore, thisinteraction would be a way to attenuate NO toxicityto the host cell. NO also interacts with catalase,another heme-protein that is critical in protectingcells against H2O2. Catalase inhibition leads toincreased levels of H2O2 because of reversibleinhibition of H2O2 breakdown.

42 Treatment seemsto inhibit catalase activity inside peroxisomes.Besides increased NAD(P)H oxidase activity, theelevated levels of H2O2 found inside vesiclescan also be a consequence of this catalase

inhibition by NO.We have shown that macrophages triggered with

Canova have an increase in their activity of NAD(P)Hoxidase as well as that of iNOS, consequentlyproducing ROS and NO respectively. Phagocyteanti-microbial mechanisms often work synergisti-cally. ROS are very unstable, as they posses one ormore unpaired electrons which can make themhighly reactive. NO reacts very rapidly with oxygenradicals.43 The chemical and biological interactionof NO and ROS with various biological molecules hasimportant consequences in the mechanisms of

different immunological and pathological con-ditions. Macrophages have the opportunity toproduce OK2 and NO in nearly equimolar amounts.As NO migrates near to the source of OK2 , it reacts toform peroxynitrite (ONOOK). Thus the primarychemistry of ONOOK would be within closeproximity of the OK2 source.

28,36 Activation ofNAD(P)H oxidase leads to OK2 increase as well asH2O2 inside vesicles (Fig. 3(C) and (D)). iNOSexpression was seen near vesicle sites (Fig. 2(C)and (D)). As NO is small and uncharged, it cantraverse the vesicle membrane8 and we can assumethat in macrophages treated with Canova ONOOK

formation would be occurring within vesicles. It canbe supported by the fact that OK2 release fromtreated cells diminished considerably after 15 min(Fig. A and B). Reduced cytochrome c can bereoxidized by oxidants such as ONOOK, diminishingapparent rates of cytochrome c reduction.44

ONOOK is not only a free radical, but is a short-lived and far more reactive species than itsprecursors. Its highly reactivity with enzymes,macromolecules and lipids have been shown toinfluence cellular functions. Formation of ONOOK

from OK2 and NO may be useful in some situations.Phagocytes can generate ONOOK to help in killingpathogens. It can also act, in some circumstances,as an anti-oxidant defence by preventing anincrease in the concentration of OK2 and H2O2.ONOOK also oxidizes and nitrates a variety ofbiological targets and is a potential mediator of

cytotoxic effects of nitric oxide.4547

Canova is probably destined to control theRNS/ROS balance therefore playing an importantrole in the immune response. Its action leading tomacrophage activation can be considered as abiological response modifier, as it provides animmune modulatory response directed to enhancethe individuals own immunity to favor a particularimmunological response.48 The precise pathwayinduced by CA is still unknown, but speculationsmay be suggested. The NF-kB/Rel is a family oftranscriptional factors that regulate the expressionof numerous cellular genes and to play important

roles in immune and stress responses, inflammationand apoptosis. It has been suggested that intra-cellular ROS levels up-regulate activity of the NF-kB/Rel family.38 It is known that up-regulation ofiNOS expression9 and a selective repression of TNFatranscription49 is due to NF-kB (p50 dimers). Basedon our findings, the most likely mechanism that canaccount for the biological effects of CA onimmunomodulation probably involves the acti-vation of the NF-kB/Rel family. However it deservesfurther investigation

In conclusion, our findings provide a possible

explanation on how treatment with Canova mayenhance immune functions, which could be par-ticularly important in the cytotoxic actions ofmacrophages. It can be considered as a newadjuvant therapeutic approach to known therapies.

Acknowledgements

The authors thank PARANA TECNOLOGIA, CAPESand PIBIC/CNPq for financial support. We aregrateful to Dr Vanette Thomaz Soccol for providing

L. amazonensis; Dr Samuel Goldenberg, IBMP forT. cruzi.

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