Original Research Article

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1 IUBMB Life, 48: , 1999 Copyright c 1999 IUBMB /99 $ Original Research Article Lucigenin Luminescence Elicited by Microsomes and Its Modulation by Nitroazole Compounds I A Schepetkin Institute of Oncology, Siberian Branch of the Russian Academy of Medical Sciences, Tomsk, Russia Summary The lucigenin luminescence elicited by rat liver microsomes and its modulation by the nitroazole compounds metronidazole and sanazole (drug AK-2123), as well as the rates of lucigenindependent NADPH consumption and cytochrome c reduction, were studied The obtained data suggest that the luminescence can be the result of univalent lucigenin reduction by microsomal NAD(P)Hreductases, generation of superoxide anion radical in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of cytochrome P450 heme on dioxetane decomposition, followed by light emission IUBMB Life, 48: , 1999 Keywords Cytochrome P450; lucigenin; luminescence; metronidazole; microsomes; sanazole INTRODUCTION Lucigenin (bis-n-methylacridinium) (Luc 2+ ) has been used frequently for luminescence detection of superoxide anion radical (O 2 ) in various biological systems (1 12) Now these results must be revised in connection with data obtained for the mechanism of Luc 2+ luminescence in enzyme (reductase) systems, which may be summarized in the form of the following scheme (13): Luc 2+ + EnzH 2! EnzH + LucH + [1] LucH + + EnzH! LucH 2 + Enz [2] LucH + + LucH +! LucH 2 + Luc 2+ [3] LucH + + O 2! Luc 2+ + O 2 + H + [4] Received 29 April 1999; revised 15 June 1999; accepted 19 July 1999 Address correspondence to Igor Schepetkin Fax: (007) oncology@infotsuru 1 Abbreviations: Cyt c 3+, cytochrome c; Luc 2+, lucigenin; SOD, superoxide dismutase LucH + + O 2! dioxetane [5] Dioxetane! light + 2 acridone [6] Luc 2+ is reduced univalently to LucH + by nitric oxide synthase, xanthine oxidase, lipoamide dehydrogenase, glucose oxidase, aldehyde oxidase, and NADPH-cytochrome P450 reductase (13 15) O 2 reacts with LucH + to yield an unstable dioxetane (reaction 5), which decays by a light emission (reaction 6) However, in the microsomal system the mechanism of Luc 2+ luminescence has not been studied enough Barth et al (16), discussing their results of activation Luc 2+ luminescence by drugs with antioxidative properties, eg, denbufylline and nabumetone, from the position that Luc 2+ luminescence tests the basal O 2 production in the microsomes, concluded that lipid peroxidation and reactive oxygen species generation are not linked absolutely Later Klinger et al (4) demonstrated that both catalase and superoxide dismutase (SOD) negligibly inhibited Luc 2+ luminescence in the microsomal system and suggested that predominantly hydroxyl radical determines this type of luminescence This supposition, however, seems unlikely, given the considerable role of H 2 O 2 in forming hydroxyl radical in microsomes (17) In the present study Luc 2+ luminescence elicited by rat liver microsomes and its modulation by the nitroazole compounds sanazole and metronidazole are investigated The roles in this process of a avoprotein domain and of a heme-containing oxidase domain of cytochrome P450 are discussed EXPERIMENTAL PROCEDURES SOD, bovine milk xanthine oxidase, horse heart cytochrome c, and salts for buffer solution (NaH 2 PO 4 and Na 2 HPO 4 ) were obtained from Sigma Catalase, Luc 2+, NADPH, xanthine, and glycerol were from Serva Sodium azide (NaN 3 ) and phenobarbital were from Merck, NADH was from Reanal, and metronidazole (2-methyl-5-nitroimidazole-1-ethanol) was from Unique Sanazole [drug AK-2123; N-(2 0 -methoxyethyl)-2-( nitro triazolyl)acetamide] was kindly given by Dr T Kagiya (Health 499

2 500 SCHEPETKIN Research Foundation, Kyoto, Japan) Sodium phosphate buffer (01 M, ph 74), prepared with doubly distilled water, was used to prepare all other solutions Carbon monoxide (CO) was prepared by reacting formic acid with concentrated sulfuric acid at 100 ± C For cytochrome P450 (CYP2B) induction, the rats (Wistar, males) received three daily doses of phenobarbital, 80 mg/kg of body weight, intraperitoneally and were killed 24 h later The liver homogenate was centrifuged at 9,000g for 30 min, and the resulting supernate was then ultracentrifuged at 105,000g for 60 min The pellet of microsomes obtained was resuspended in phosphate buffer with 20% glycerol, stored at 50 ± C and then thawed and diluted immediately before use Microsomal protein was determined by the biuret reaction NADPH consumption (k = 340 nm, e = 622 mm 1 cm 1 ) and cytochrome c (Cyt c 3+ ) reduction (k = 550 nm, e = 185 mm 1 cm 1 ) were monitored spectrophotometrically with the Specord M40 (Carl Zeiss, Germany) device K m and V max values obtained with Luc 2+ concentrations of 15 to 200 l M were calculated by Hofstee plots (18) Light emission was monitored with a Model 1251 luminometer (LKB, Sweden) All measurements were made at 25 ± C Each effect was tested in at least three independent experiments RESULTS AND DISCUSSION In adding NADPH to an incubation medium containing microsomes, negligible consumption of NADPH was observed However, the combination of Luc 2+, microsomes, and NADPH caused complete oxidation of NADPH within 30 min and an intense luminescence (Fig 1) At NADPH concentrations from Figure 1 NADPH-dependent Luc 2+ luminescence elicited by microsomes (1) and effect of Luc 2+ on the NADPH consumption by microsomes (2) Reaction mixtures contained 115 l M NADPH, and reactions were initiated with 220 l g/ml of microsomal protein As indicated by arrows, Luc 2+ was added (40 l M nal concentration) at 10 min Figure 2 NADH-dependent Luc 2+ luminescence of microsomes Reaction mixtures contained 40 l M Luc 2+ and 220 l g/ml of microsomal protein Reactions were initiated by adding at 10 min NADH: 15 l M (curve 1), 60 l M (curves 2 and 5), 250 l M (curve 3), and 500 l M (curve 4); curve 5, SOD (20 U/ml) was added at 16 min 60 l M to 1 mm, the peak magnitude increased but did not reach a plateau pattern Including catalase (1000 U/ml) in the reaction mixture inhibited integral (for 30 min) luminescence by 7%, whereas including NaN 3 (10 mm) inhibited it by 20%, SOD (20 U/ml) by 30% When NADH was used in the concentration range of 60 to 250 l M as electron donor, luminescence attained its maximum for 20 5 min and remained high before all of the NADH or Luc 2+ was consumed (Fig 2) Catalase (1000 U/ml) did not inhibit this luminescence, and NaN 3 (10 mm) decreased integral (for 30 min) luminescence by 12% SOD (20 U/ml) decreased peak luminescence by 20% but prolonged the period of light emission (Fig 2, curve 5) This can be connected with the decrease of LucH + consumption in reaction 5 and dioxetane formation in other pathway Luc 2+ also increased the rate of cytochrome c (Cyt c 3+ ) reduction in microsomal suspension in the presence of NADPH, but this reaction was only partially inhibited by SOD (data not shown) These data support the reaction: LucH + + Cyt c 3+! Luc 2+ + H + + Cyt c 2+ [7] which was demonstrated in the systems of NADH/xanthine oxidase and glucose/glucose oxidase (13) The formation of Cyt c 2+ can testify to the rate of Luc 2+ univalent reduction in case of Cyt c 3+ application insaturating concentrations in normoxygenic

3 LUCIGENIN LUMINESCENCE ELICITED BY MICROSOMES 501 conditions In this case the rate of reactions 2 and 3 is negligible, and Cyt c 3+ is reduced by LucH +, O 2, or both Apparent K m values for Luc 2+ (Km Luc ) were 36 and 50 l M in normoxygenic media in reactions of Luc 2+ -dependent NADPH oxidation and Cyt c 3+ reduction, respectively Higher values for K m Luc determined by the rate of Cyt c3+ reduction in media may testify to the formation of dioxetane in reaction 5 and the two-electron reduction of Luc 2+ in reactions 2 and 3 The maximal intensity of NADPH-dependen t luminescence was registered at Luc 2+ concentrations close to the mean Km Luc Increasing the concentration of Luc 2+ to 100 l M was followed by a decrease in the luminescence intensity (data not shown), perhaps because of absorption by Luc 2+ of light emitted by electronically excited N -methyl acridone The peak structure of the curves for NADPH-dependen t luminescence of Luc 2+ in comparison with the plateau curves in the presence of NADH may be connected with the participation of reduced (by NADPH) cytochrome P450 in the formation and decay of dioxetane Indeed, under aerobic conditions, NADH is not able to keep cytochrome P450 in the reduced state because of the latter s high rate of autooxidation (19) A partially inhibiting in uence of SOD and catalase on Luc 2+ luminescence of microsomes shows that luminescence seems to result not only from O 2 generation (reaction 4) and the subsequent reaction O 2 with LucH + to form dioxetane (reaction 5) but also from the formation dioxetane in the heme-containing oxidase domain of cytochrome P450 The latter pathway supposes the formation of dioxetane irrespective of free O 2 and accounts for 70% of luminescence (luminescence not inhibited by SOD) The presence of the complex CO-reduced hemoprotein was investigated for further studying the role of heme-containing domain of cytochrome P450 in Luc 2+ luminescence The addition of Luc 2+ to microsomes containing the CO-reduced (by NADPH) hemoprotein complex was followed by the disappearance of carboxy-speci c absorption spectrum (Fig 3) as well as by an increase in luminescence intensity of 20 80% versus the control (without complex), depending on the duration (from 20 to 60 s) of exposing the microsomal suspension to CO (data not shown) The increase in luminescence intensity after exposing the microsomal suspension to CO may be explained by reservation of the reduced form of cytochrome P450, which is released from the complex CO-P450 in adding Luc 2+ and participates in (a) the enzymatic formation of dioxetane and (b) the decomposition of dioxetane The process of introducing molecular oxygen to Luc 2+ on two-component enzyme complex cytochrome P450 may be represented by the following hypothetical scheme: Luc 2+ e! LucH + e, O 2! dioxetane! light + 2 acridone [8] Light emission in the course of dioxetane decay can take place both on cytochrome P450 indirectly and after dissociation of dioxetane and cytochrome P450 In the rst case, the quantum Figure 3 CO-speci c absorption spectrum of microsomal hemoprotein in the absence (1) or presence (2) of Luc 2+ In this experiment, microsomal protein (300 l g/ml) was present in the sample and reference cuvettes of the spectrophotometer and was reduced with 150 l M NADPH for 2 min Then CO was blown into the sample cuvette for 1 min and specrophotometric analysis was completed after adding or not adding Luc 2+ (65 l M) to the sample and reference cuvettes yield of luminescence will be negligible because of nonradiative energy transfer to a heme of cytochrome P450 Agents with different electron-acceptor properties or relationships to the components of cytochrome P450 can signi - cantly change the contribution of reactions 5 and 8 to the formation of dioxetane and thereby modulate the total output of light emission Therefore, for further study of the mechanism of the process, the nitroazole compounds metronidazole and sanazole were added to the system examined Both Luc 2+ and sanazole increase the rate of Cyt c 3+ reduction in the enzyme system xanthine/xanthine oxidase (20) and by microsomes when NADPH is used as an electron donor (Fig 4), whereas metronidazole has no effect This can be understood by the greater capacity of sanazole and lucigenin (but not metronidazole) to accept electrons in the active centre of avin reductases and their transfer to Cyt c 3+ The addition of sanazole to microsomes caused a considerable inhibition of luminescence, whether NADPH or NADH was used as the electron donor After preliminary exposure of the microsomal suspension to CO, the inhibiting action of sanazole was manifested to a lesser degree Concentrations of sanazole of > 1 mm in the presence of NaN 3 (10 mm) or SOD (20 U/ml) inhibited luminescence almost completely In contrast, metronidazole caused an increase of light emission when NADPH was used as the electron donor but did not depend on this process when NADH was applied (Fig 5) Sanazole strongly inhibited luminescence in the xanthine/ xanthine oxidase enzyme system The inhibiting action of metronidazole on luminescence in this system was considerably

4 502 SCHEPETKIN Figure 4 Effect of metronidazole, sanazole, and Luc 2+ on rate of Cyt c 3+ reduction by microsomes Reaction mixtures contained 100 l M NADPH and 40 l M Cyt c 3+ Reactions were initiated with microsomal protein, 200 l g/ml Where indicated with arrows, metronidazole (3 mm), sanazole (3 mm), and Luc 2+ (60 l M) were added less (Fig 6), in agreement with the results of other investigators (21) Neither sanazole nor metronidazole absorb light in the eld of light emission of Luc 2+ (data not shown) Thus, the inhibition action of sanazole on Luc 2+ luminescence is comparable in two systems, ie, microsomal NAD(P)Hcytochrome P450 reductase/cytochrome P450 and xanthine/ xanthine oxidase, and can be connected with competition of sanazole (with regard to Luc 2+ ) for electrons in the active centre of avin reductases On the other hand, the relationship of metronidazole to a heme-containing oxidase domain of cytochrome P450 (22) can cause the activating effect of this agent on luminescence, maybe as a result of acceleration of cytochrome P450 and dioxetane dissociation, which decreases the nonradiative energy transfer to a heme of cytochrome P450 at dioxetane decay A comparison of the ratio of intensities of Luc 2+ luminescence to luminol luminescnce in various biological systems shows very high values of this index for microsomes (4, 5, 23 26) (Table 1) Apparently, luminol luminescence more adequately re ects a basal level of reactive oxygen species in microsomal suspension In fact, SOD and catalase strongly inhibit (> 90%) luminol luminescence of microsomes (4, 27) On the contrary, Luc 2+ luminescence is caused by enzyme univalent reduction of Luc 2+ and O 2 generation in redox-cycling of LucH + Generation of O 2 explains an increase of O2 consumption in adding Luc 2+ to microsomes of the smooth muscular tissue of the pulmonary artery in a calf (10) The presence of such a redox cycle under aerobic conditions and an inrease in O 2 consumption is considered to be a common property for some Figure 5 Effect of metronidazole and sanazole on Luc 2+ luminescence in microsomal suspension: NADPH-dependent luminescence in the presence of metronidazole (1), sanazole and CO-reduced hemoprotein (before addition of Luc 2+ and sanazole, CO was bubbled through the microsomal suspension for 1 min) (4), sanazole (5), sanazole and 10 mm NaN 3 (6), sanazole and 20 U/ml SOD (7) NADH-dependent Luc 2+ luminescence in the presence of metronidazole (2) and sanazole (3) Reaction mixtures contained 60 l M Luc 2+, microsomal protein (220 l g/ml), the indicated concentration of nitroazole compound (metronidazole or sanazole), and 25 l M NADPH or 250 l M NADH The effect was estimated as a magnitude of the ratio of integral (for 30 min) luminescence value treated with nitroazole compound to that of the control (with no agents nitroazole, SOD, or NaN 3 and no CO treatment); data are averages of three experiments electron-acceptor compounds subject to univalent reduction by microsomal NAD(P)H-reductases (28) On the other hand, metabolism of Luc 2+ on the enzyme complex NAD(P)H-cytochrom e P450 reductase/cytochrome P450 Table 1 The ratio of intensities of Luc 2+ (I luc ) to luminol (I lum ) luminescence in various biological systems Biological system I luc / I lum C luc / C lum a References Human neutrophils , 24 Human monocytes < Mouse neutrophils Mouse macrophages Rat liver microsomes Rat liver microsomes + Fe a Ratio of molar concentrations of Luc 2+ (C luc ) and luminol (C lum )

5 LUCIGENIN LUMINESCENCE ELICITED BY MICROSOMES 503 Figure 6 Effect of metronidazole (1) and sanazole (2) on Luc 2+ luminescence in enzyme system xanthine/xanthine oxidase Reaction mixtures contained 100 l M xanthine, 60 l M Luc 2+, the indicated concentration of nitroazole compound (metronidazole or sanazole), and 10 mu/ml xanthine oxidase The effect was estimated as a magnitude of the ratio of integral (for 20 min) luminescence for the sample treated with nitroazole compound to that for the control (no drug treatment); data are averages of three experiments apparently may have much in common with bioluminescence reactions, similar to luciferin conversion in the bacterial complex NAD(P)H-FMN oxidoreductase/luciferase One must take into account the possibility of activating luminophores and, in particular, the analogs of luciferin and laser dyes (29) by the components of cytochrome P450 when searching for new probes to register the processes of lipid peroxidation and generation of 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