Gen Physiol Biophys (1997), 16, 3—14 3

Effect of Phenothiazines on Activated Macrophage-Induced Luminol-Dependent Chemiluminescence

T T R A Y K O V , V H A D J I M I T O V A , P G O L I Y S K Y AND S R I B A R O V

Department of Biophysics Sofia University School of Medicine Sofia, Bulgaria

Abstrac t . The inhibitory effect of some phenothiazme neuroleptics (chloipro-mazine, levomepiomazine, thioridazine, piomethazme and tiifluoperazme) on the ability of rat peritoneal maciophages to produce 0 7 during phagocytosis was in­vestigated The superoxide íadical release was estimated by measuiing the lummol-dependent chemiluminescence (CL) The effect of diugs was studied m the coricen-tiation íange of 0 1-100 /miol/1

Additional experiments to determine the ability of the diugs to scavenge O 7 were earned out They included measuiing the effect of phenothiazines on the luminol-dependent CL in systems with enzymatically (xanthine-xanthine oxidase) and non-enzymatically (K02) generated 0 7 The ability of phenothiazines to scav­enge 0 7 was additionally tested by a "non-lummescence" method m which the superoxide concentration was determined spectiophotometrically by the i eduction of nitro blue tetiazohum to formazan

All diugs tested decreased significantly CL of stimulated macrophages at con­centrations greatei than 1 //mol/1 The C50 values were between 0 45 and 1 74 /imol/1

Also phenothiazines were found to act as scavengers of 0 7 Howevei, this effect occurred at significantly highei diug concentrations The C50 values for 50% scavenging of O 7 111 systems with different souices of O7 were in the concentration íange of 5-160 /^mol/1

These results suggested that phenothiazines piedommantly affected the ability of macrophages to produce O7 during phagocytosis The findings may provide some insight into the untoward effects of the diugs tested

Key words: Chemiluminescence - Free radicals Macrophages - Phenothiazines - Supeioxide

Correspondence to Prof Dr Stefan R Ribaiov, Dept Biophysics, Sofia Univeisity of Medicine, 2 Zdravé Str , Sofia 1431, Bulgaria, Phone/Fax +359-2-541-372 E-mail physicma@bgcict acad bg

4 Traykov et al

Abbreviations: 0 7 supeioxide íadical, K0 2 potassium superoxide CL chemiluminescence C50 - drug concentiation which causes 50% reduction of the paiametei leflectmg the concentiation of 0 7 m the system PMNLs polymor phonucleai leukocytes, CPZ chloipromazine, LVPZ - levomepiomazine TRDZ thioiidazine PMZ piomethazme, TFPZ tufluopeiazme, PBS phosphate buffer solution HBSS Hanks balanced salt solution, OZ opsonized zymosan CL-I chemiluminescence index CL SI chemiluminescence scavenging index SPh SI spectiophotometnc scavenging index LP - lipid peioxidation

Introduction

It is well known (Johnston et al 1978) that macrophages activated by vaiious stimuli íelease íeactive oxygen species, such as superoxide radical hydiogen pei-oxide, etc which are thought to be majoi effe( toi molecules mediating the killing of miciooigamsms (Nathan et al 1979) On the othei hand in piachcal medicine tieatment with different diugs could consideiably affect the maciophages' ability to generate icactive oxygen species, and consequently then bactericide activity (Dunckei and tollman 1986 Itoh et al 1989) Phenothia/ine denvatives aie widely used m contempoiaiy medicine (Baldessarmi 1992) and have been extensively in vestigated Theie is limited evidence foi then potential to modulate the geneiation of icactive oxygen species by phagocytes Tamguchi et al (1988) lepoited that tufluopeiazme and chloipiomazme markedly inhibited superoxide production of íabbit PMNLs at concentíations higher than 10 //mol/1 Also it has been demon-stiated (Keldei et al 1991) that chloipiomazme inhibited the íespnatoiy burst of human PMNLs as íecoided by lucigemn dependent chemiluminescence

In all these investigations, however supeioxide pioduc tion was deteimined by nieasuiing the superoxide concentiation in the system This is coirect foi systems only m which superoxide, once released, does not mteiact with the chug tested

Theie was no diiect evidence that phenothiazines are scavengeis of O7 How-evei, it has been shown that chloi promazine inhibited lipid peioxidation m lipo­somes (Bmdoli et al 1988), íat synaptosomes (Siam and Bmkova 1992) and hvei miciosomes (Rider et al 1988) Since the initiation of lipid peioxidation needs an mcieased pioduction of 0 7 , these íesults seem to imply that chlorpiomazme (and possibly also othei phenothiazines) is able to scavenge 0 2

In the context of these data, the aim of the present woik was to test the possibility of phenothiazines scavenging 0 7 generated m a system of activated maciophages This investigation enabled us to estimate the íeal modulation effect of phenothia/mes on the ability of macrophages to pioduce OJ

Phenothiazines and Chemiluminescence of Macrophages 5

Mater ia l s and M e t h o d s

Chemicals and preparations

The following phenothiazines were studied chlorpiomazme, levomepiomazme, thio ndazine, piomethazme and tiifliioperazme Chloipiomazme, thioiidazine piomet hazme hydrochlorides and tnfluoperazme chhydrochloiide weie obtained fioni Sig­ma (St Louis, MO, USA) Levomepromazme (2-methoxy-10-[3-dimethylamino-2-methylpiopylj-phenothiazme) hydiochlonde was kmdlj donated by Pha imaclnm Co (Sofia, Bulgaria) Zymosan was purchased fiom Serva Feinbiochemical (Hei-delbeig, Geimany) Potassium supeioxide ( K 0 2 ) and most of the othei leagents were obtained from Fluka Chémie AG (Buchs, Switzeiland), and weie of finest grade

Hanks ' balanced salt solution without phenol led and bicaibonate (Sigma) was dissolved in ledistilled watei to 290 mOsm and the pH value was adjusted to 7 4 with chsodium phosphate

The chemiluminescence íeagent was piepaied by dissolving lummol m a small amount of 0 01 mol/1 NaOH Then the solution was diluted with 50 nimol/1 PBS to give lummol concentiation of 1 mmol/1, and the pH was adjusted to 7 4 with 0 01 mol/1 HC1

Diugs weie dissolved m PBS pH 7 4 Then final concentiations m the samples investigated aie given m the figure legends

K 0 2 was dissolved m anhychous dimethyl sulfoxide (AldiRli-Chemie Stem-heim, Geimany) to concentiation of 1 nimol/1 The solution was stoied undei m-tiogen and was used not later than 2 houis aftei the piepaiat ion

Opsonized zymosan was prepaied accoiding to Allen (1986), and suspended m HBSS to concentiation of 20 mg/ml

Preparation of peritoneal macrophage suspension

Male albino Wistai íats (180 200 g) weie used The lats weie injected mtia-peiitoneally with 20 ml HBSS Aftei 1 h the lemammg solution was sucked out from the pentoneal cavity The led blood cells weie lemoved bv a bnef (20 seconds) osmotic shock The peritoneal exudate cells weie washed 3 times m HBSS, tested foi viability with tiypan-solution (4 g/1) and then lesuspended to yield of 5 x 10b

cells/ml

Methods

OJ was registered by measuring the lummol-dependent CL (Allen and Loose 1976 Itoh et al 1989) For this piupose, LKB 1251 lummometei (Biooibit Tuiku Fin­land) set at 37°C was used It was connected to an AT-type computei via seiial mteiface, and MultiUse piogiam vei 1 08 (Biooibit) was used to collect da ta

6 Traykov et al

Thiee types of CL assays weie used A s s a y I (lummol dependent CL of OZ stimulated macrophages)

In this case, the sample cuvette contained 100 /d lummol solution, 600 /A HBSS 100 /ň diug solution (100 //l HBSS for controls), and 100 //l cell suspension Each sample (pH 7 4) was incubated for 10 mm Then, 100 /xl of OZ weie added using a piogiammed automatic dispensei and CL was íegistered foi 40 m m A s s a y I I (lummol dependent CL in a system of xanthine-xanthine oxidase gene­rated superoxide)

One ml PBS pH 7 4 containing 0 1 mmol/1 lummol, 1 mmol/1 xanthine and the d ing at concentiations as indicated in the figuie legends weie used In the control sample diug was omitted Each sample was incubated for 10 mm at 37°C Then 20 //l of xanthine oxidase (100 IU/1 m PBS) were added Subsequently the CL signal was measuied for 5 mm A s s a y I I I (lummol-dependent CL m a system of K 0 2 - p r o d u c e d superoxide)

The assa\ was earned out using 1 ml samples of P B S pH 7 4, containing 0 1 mmol/1 lummol and the chug (in contiol sample drug was omitted) The CL was measuied immediately aftei addition of 20 /xl KO2 solution The íelease of supei oxide in this case is a fast piocess Therefoie CL was measured using the 'flash assay" option of the MultiUse piogiam, eveiy 50 milliseconds To get some addi tional mfoimation, chugs weie tested by the following 11011 luminescence method A s s a y I V (spectiophotometiic íegistration of 0 2 geneiated by K 0 2 )

The mhibitoiy effect of the tested diugs on the leduction of NBT to formazan m a system of K0 2 -geneia ted 0 7 was studied Foi this puipose, we added 20 /d K 0 2 solution to 1 ml of 50 mmol/1 P B S , pH 7 4 containing 0 04 mmol/1 N B T and the tested diugs The leaction mixtuie was vigorously mixed aftei the addition of K 0 2 solution and the absorbance at 560 nm was measured using a Shimadzu UV 260 spectiophotometei Assays weie íepeated five times foi each chug and contiol

Statistics

Foi multiple gioup compansons, one way analysis of variance (ANOVA) was em ployed followed by Bonfenoni 's test for t iuly significant drffeiences Statistical sig­nificance was defined at P < 0 05 The statistical proceduies were peiformed with G i a p h P a d InStat software, version 2 04, USA Data aie expressed as mean ± S D

R e s u l t s

In oui initial expenments, we studied the effect of drugs on the CL in a system of zymosan activated pentoneal macrophages (assay I) T h e íatio (111 percentage) between CL in the piesence and 111 the absence of the tested drugs was termed CL-I (CL index) (Fig 1) As is well seen, at concentiations highei than 1 /imol/1 all diugs maikedly deciease the lummol-dependent CL in such a system At drug

Phenothiazines and Chemiluminescence of Maciophages 7

F i g u r e 1. Deciease of 1 he himinol-dependenl CL of p< u t o n e a l mac rophages in the pies ence of phenotlna/ines (assay I) T h e sample cuvette contained 100 /tl lummol solution (1 mmol/1) 600 itl HBSS (290 m O s m pH 7 1) 100 i/l drug solution or 100 //l HBSS in contiol and 100 /d cell suspension (5 x 10Ď tells/ml) After addit ion of 100 //l OZ (20 m{,/ml) the chemihiminescence was lecoided foi 10 m m T h e ratio (in peicentages) of CL in the presence and m the absence of the tested drugs was termed C ľ I

concentiation of 0 1 mmol/1 the CL-I was in the íange of 0 7 3'/ It is well known (Allen and Loose 1976) that lunimol-dependtnt chemilumi­

nescence leflects the concentiation of supeioxide lachcals m the leaction mixture Theiefoie it may be suggested that phenothiazines somehow deciease the 0 2 con­tent in the system of activated maciophages At least two diffeient mec hanisms may be involved drug induced changes of the ability of maciophages to pioduce 0 7 d u n n g phagocytosis (leal modulation effect of the diugs on the bactencidal activ­ity of the macrophages), and/or chug induced scavenge of 0 2 geneiated Initially, we studied the 07-scavengmg effect of phenothiazines The effects of chugs on the lummol-dependent CL in a system with enzymatic ally geneiated O J (assay II) aie shown m Fig 2 We believed that the CL íatio in the piesence and m the absence of the tested diugs m this system would íeflect the 07-scavengmg piopeities of the diugs Theiefoie, this latio was teinied CL-SI (chemiluminescence scavenging index)

It was established tha t , at concentiations above 1 //mol/1, all drugs decreased modeiately CL-SI m a xanthine-xanthine oxidase system The effect was most distinct foi chloipiomazme while being vague foi tnfluoperazme These íesults possibly mean that phenothiazines weie able to scavenge 0 7

Diug induced deciease of the lummol-dependent CL m a system of xanthine-xanthine oxidase-geneiated supeioxide íadicals may be due to the well known fact (Lullmann-Rauch and Scheid 1975, Abdalla and Bechara 1994, Motohashi 1995)

8 Traykov et al

Drug concentration (mol/1)

Figure 2. Effect of phenothiazines on the lummol-dependent CL in a system of xanthine-xanthine oxidase- generated 0 7 (assay II) The leaction mixture contained 1 ml PBS pH 7 4 0 1 mmol/1 lummol, 1 mmol/1 xanthine and diug at concentrations as indicated In the contiol sample drug was omitted After addition of 20 /d xanthine oxidase (100 IU/1 m PBS) the chemiluminescence was measured for 5 mm The ratio of CL m the presence and in the absence of the drugs was termed CL SI

that phenothiazines aie inhibitois of a numbei of enzymes Theie aie no data show­ing that xanthine oxidase is among them Neveitheless, the ability of phenothiazines to scavenge 0 7 was tested also m a system with K 0 2 geneiated 07 (assay III) In this case, the íelease of O j is an extiemely fast piocess, and CL was registered by the "flash assay" option of the MultiUse progiam every 50 milliseconds Typical time couises of the lummol-dependent CL m the system of K02-geneiated 0 7 aie shown 111 Fig 3, and the lesults obtained aie summarized 111 Fig 4 We did not find any significant changes of CL-SI below drug concentiations of 10 /tmol/1 At higher concentiations, chloi promazine, promethazine and tufluopeiazme slightly reduced CL-SI At the concentration of 0 1 mmol/1, the tested diugs decreased CL-SI 2 5 to 6 5-fold

On the othei hand, the effect of phenothiazines on the lummol-dependent CL (assays II and III) may be due, at least 111 part to interaction of drugs with excited lummol molecules and/01 with the light emitted If this weie the case, the result would be a diop m CL m the presence of the diugs, even if they were not 0 7 scavengers at all Theiefore, a "non-luminescent" study (assay IV) was earned out In this case, the concentiation of 0 7 m the system was measuied by a NBT-test The latio (in pei cent age) of the mciease of absoibance at 560 11m 111 the piesence and 111 the absence of the diug was teimed SPh-SI (spectrophotometnc scaveng­ing index), (Fig 5) The data obtained weie similar to those obtained from the "luminescent" experiments (assays II and III) The diugs had no effect below the concentiation of 1 //mol/1 Tnfluopeiazme did not cause any changes of the SPh-SI

Phenothiazines and Chemiluminescence of Macrophages 9

3

TO

-5->.

• ^

w (1)

O

0.2 0.4 0.6 Time (s)

0.8

Figure 3. Typical lummol-dependent CL time courses in a system with KC^-generated OJ (assay III) The sample cuvette contained 1 ml PBS (pH 7 4), 0 1 mmol/1 lummol and drug In control sample the drug was omitted The chemiluminescence was registered aftei addition of 20 /ti KO2 (1 mmol/1 in dimethyl sulfoxide)

5"

Č75 1

0

100

80

60

40

20

0

^ ^ C P Z Fffl3LVPZ t^^TRDZ 5583 PMZ

]TFPZ

107 106 105

Drug concentration (mol/1)

104

Figure 4. Inhibition by phenothiazines of the lummol-dependent CL m a system with K02-generated superoxide radicals by phenothiazines (assay III) The sample cuvette contained 1 ml PBS (pH 7 4), 0 1 mmol/1 lummol and drug In control sample the drug was omitted The chemiluminescence was registeied after addition of 20 /d KO2 (1 mmol/1 in dimethyl sulfoxide) The ratio of CL in the presence and in the absence of the drugs was termed CL-SI

even at 10 /mrol/1 At such concentiation, the other caused insignificant decrease in SPh-SI Fuither increase of the diugs concentiation led to well expiessed ieduction

10 Traykov et al

107 106 105 104

Drug concentration (mol/1) Figure 5. Scavenging of superoxide radicals by phenothiazines, determined by the 'non luminescent" NBT test (assay IV) The sample cuvette contained 1 ml of 50 mmol/1 PBS (pH 7 4) 0 04 mmol/1 NBT and drug After addition of 20 /d KO2 (1 mmol/1 in dimethyl sulfoxide) the change of absorbance at 560 nm was measured The ratio (in percentage) of the change of absorbance at 560 nm in the presence and in the absence of the drugs was termed SPh-SI

of this index Thus, 0 1 mmol/1 chlorpiomazme or levomepromazme decreased its value appioximately 3 fold wheieas theie was a less than twofold decrease after tnfluopera7ine

Discussion

The dose-effect íelations in Figures 1,2 4 and 5 aie sigmoid for all tested drugs To compaie the íesults obtained from the assays, we therefore calculated the values of (ľ50, i e drug concentiations causing a 50% decrease of the parameteis (CL-I for assay I, CL-SI for assays II and III, and SPh-SI for assay IV) i effecting the concentiation of OJ in the respective reaction mixture (Table 1) The calculations used fitting of the data to the "sigmoid" model

C L - I(CL - SI oi SPh - SI) = 100/ [l + ioB(^c-\gCrM)j

wheie B is the coefficient (hill slope), and C is the diug concentration The results obtained in assays II, III and IV (columns 3, 4 and 5, íespectively)

clearly show that all the tested phenothiazines aie able to scavenge 0 7 Howevei, we did not find significant diffeiences between the íesults obtained Neveitheless, it seems that C 5 0 (assay II) < C 5 0 (assay III) < C50 (assay IV) These diffeiences and tendencies might be due to the diffeient íates of 0 7 generation by the used 0 7 sources Foi the xanthine - xanthine oxidase system, the results might also be influ­enced by the chug-induced deciease of the xanthine oxidase activity Fuitheimore,

Phenothiazines and Chemiluminescence of Macrophages 11

Table 1. Concentrations (C50) of phenothiazines leading to 50% decrease of the parameter reflecting the O j content m the reaction mixtures of assays I-IV The calculations were carried out by a non-linear regression analysis The correlation coefficient values R were > 0 995 which indicates a high goodness of fit and non-significant deviation from the used model

Drugs

Chloi promazine Levomepromazme Thioridazine Promethazine Tnfluopeiazine

CL-I (assay I)

1 05 ± 0 12 0 45 ± 0 01 1 74 ± 0 18 0 8 5 ± 0 11 102 ± 0 15

C50,

CL-SI (assay II)

21 70 ± 0 20 4 96 ± 1 20 7 06 ± 2 18

21 70 ± 6 20 71 50 ± 8 10

/Mmol/1

CL-SI (assay III)

25 30 ± 3 60 13 3 0 ± 4 80 12 2 0 ± 5 10 37 20 ± 8 10

107 00 ± 12 00

SPh-SI (assay IV)

44 90 ± 6 00 27 20 ± 10 80 32 40 ± 18 60 86 10 ± 2 1 50

157 00 ± 33 00

phenothiazines seem to be able to íeact with the excited lummol molecules and/or with the emitted light All these suggestions however, need furthei experimental venfications

As is well seen fiom column 2 m Table 1 (assay I), phenothiazines cause a well expiessed decrease of the lummol-dependent CL m a system of activated macro­phages In such a case, the C50 values íeside within the íange of 0 45 1 74 /tmol/l This effect seems to be due at least to two types of reactions of the diugs The first one involves inteiactions with macrophages and as a lesult, the cell ability to produce O7 during phagocytosis is reduced This is an actual modulation effect of phenothiazines on the bactencidal activity of the maciophages The second mtei-action involves scavenging of O7 aftei its geneiation by the activated maciophages

The contribution of the lattei interaction seems negligible Thus, any of the diugs have not shown 0 7 - scavenging pioperties at concentrations (0 45-1 74) /xmol/l (Figs 2, 4 and 5) In addition, as is seen fiom columns 3, 4 and 5 of Table 1, the C50 values for 50% scavenging of O7 geneiated from different souices range within 5-157 /tmol/1 Therefore, the C50 values for the system of activated maciophages seems to be 10-100 times smaller than the C50 values for systems with xanthine-xanthine oxidase or K0 2 souices of O7

The conclusion that phenothiazines at miciomolar concentiation may inhibit the ability of macrophage to produce OJ duimg phagocytosis is in good agree­ment with the data obtained by other investigators Tamguchi et al (1988) found that tufluopeiazme and chloipiomazme, at concentiations higher than 10 /miol/1, markedly inhibit superoxide pioduction of íabbit PMNLs Although their ob­ject (rabbit PMNLs), stimulus (chemotactic peptide n-foimyl-methionyl-leucyl-

12 Traykov et al

phenylalanine) and method for determining superoxide generation (ferncytochrome c leduction) were different from ours, then results aie similai At concentiation of 0 1 mmol/1, they found zeio supeioxide production foi both diugs Similar to ouis are also the results of Kelder et al (1991) They found that chloipiomazme inhibits the íespnatoiy burst of human PMNLs measuied with lucigenin-dependent CL They speculated that the inhibition was most piobably due to some mteifeience with processes m the cell, leading to a respiiatory buist íathei than to scavenging of the oxygen íadicals generated that provoke luminescence Oui results show that the scavenging piopeities of phenothiazines aie not negligible and must be con sidered when doing investigations with supeioxide souices m the presence of these drugs Theie has been a numbei of such studies published íecently (Dijkstia et al 1985, Wolff 1986, Tamguchi et al 1988, Kelder et al 1991) m which, howevei, possible scavenging and quenching pioperties of the drugs tested were not taken into account

So fai, we have not heaid of investigations of modulation capabilities of levo­mepromazme, pionietliazme and thioiidazme on the respiratoiy buist It could be suggested that they have similai piopeities as chloi promazine and tufluopeiazme but this would not be quite accuiate Thus it was shown (Relder et al 1991) that phenothiazines with a similar structure (chloipromazine and chlorpromazine sulfoxide) have completely different modulation capabilities CPZ strongly inhibited the respiratory burst whereas chlorpiomazme sulfoxide did not affect it at all Theiefoie íesults foi levomepiomazme, promethazine and thioiidazme obtained m oui expenments aie almost as much novel as those established by othei authors (Tamguchi et al 1988, Keldei et al 1991) foi chloi promazine and tufluopeiazme

We could not identify papeis which would repoit on the ability of pheno thiazines to scavenge supeioxide íadical Chloi promazine has been shown to inhibit moderately Fe2+/ascoibate catalyzed LP in liposomes (Bmdoli et al 1988) and íat synaptosomes (Sram and Binkova 1992) foi concentiations greatei than 10 //mol/1 Since initiation and development of LP involve reactive oxygen species these results would suggest that chloipiomazme is able to scavenge them Howevei, another explanation is also possible It is well known (Slatei et al 1985, Halhwell 1990) that phenothiazines aie able to bind non ions Obviously, such a reaction could also lead to a drop m LP, even when the drug is not a scavengei Oui íesults confiimed that phenothiazines have scavenging capabilities

The effect íepoited heiem was obtained foi an m intro system So fai, we can not say if it takes place in vivo If it does, these íesults could be important foi prac­tical medicine Foi a numbei of phenothiazines we found a sigmoid dose-dependent influence on the CL of stimulated maciophages with C5o within the íange 0 47-1 74 //mol/1 On the other hand, the theiapeutic plasma concentiations of these diugs aie 0 05-5 //mol/1 (Dahl and Strandjord 1977, Benet and Williams 1992, Krushkov and Lambev 1993) Theiefoie, the íesults may give hints foi untowaid

Phenothiazines and Chemiluminescence of Macrophages 13

effects of phenothiazme neuroleptics (Baldessaimi 1992 Holhster 1992 Kiushkov and Lambev 1993) which may be inipoitant in the theiapy of immunocompiomized patients Anothei important question is the antioxidant action of these drugs in vivo The scavenging effect established heiein is not stiong enough foi therapeu tic plasma concentiations, but the ability of lipophilic chugs to concentiate within hydiophobic regions (such as membiane mteiior) must not be lgnoied

The mechanism by which phenothiazines modulate the ability of maciophages to pioduce O7 during phagocytosis is still not cleai It may involve chug-induced inhibition of calmodulin functions (Cuinutte et al 1984, Monmoto et al 1986) Futuie studies that aie undeiway will examine these possibilities

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14 Tiaykov et al

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Final veision accepted lanuaiv 16 1997