Pharmacology & Toxicology 1992, 70, 40741 1.

Inhibition of Cumene Hydroperoxide-Induced Lipid Peroxidation by a Novel Pyridoindole Antioxidant in

Rat Liver Mcrosomes Milan Stefek, Maria Masarykova and Ludek Benes

Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dubravska cesta, 842 16 Bratislava, Czechoslovakia

(Received August 8, 1991; Accepted November 13, 1991)

Abstract: The ability of stobadine, a novel pyridoindole antioxidant, to inhibit lipid peroxidation induced by cumene hydroperoxide was investigated in rat liver microsomes. In the micromolar range stobadine effectively inhibited lipid peroxidation as measured by the formation of thiobarbituric acid reactive products. The peroxidation-related degradation of microsomal cytochrome P-450 was prevented by stobadine in the same pattern. Another line of evidence in support of the antioxidant action of stobadine was given by its inhibition of cumene hydroperoxide-induced oxygen consumption in microsomal incubations. Inhibition of lipid peroxidation was not a function of decreased bioactivation of cumene hydroperoxide, as stobadine did not affect the rate of cytochrome P-450 dependent cleavage of cumene hydroperoxide. Neither had stobadine any effect on cytochrome P-450 peroxidase function characterized by the rate of cumene hydroperox- ide-dependent oxidation of TMPD, and no direct spectral interaction with microsomal cytochrome P-450 was observed in the micromolar region. We suggest that it is the ability of stobadine to scavenge alkoxyl and peroxyl radicals that is predominantly responsible for the observed antioxidant effect.

Stobadine, ( - )-cis - 2,8 - dimethyl - 2,3,4,4a,5,9b - hexahydro - 1 H-pyrido[4,3-b]indole (fig. l), a new drug with pyridoindo- le structure, was found to exhibit antiarrhythmic and protec- tive antihypoxic effects in the myocardium (Stolc et al. 1985) and to decrease the extent of lipid peroxidation in ischaemic- reperfused brain tissue (Stolc & Horakova 1988; Horakova et al. 1990) and in phosphatidylcholine liposomes (Ondrias et al. 1989). Spin trapping with ESR technique proved stob- adine to be a potent scavenger of hydroxyl radicals (Ondrias et al. 1989; Stasko et al. 1990). Reactive oxygen species were found to be involved in cytochrome P-450 mediated rat liver microsomal biotransfomation of stobadine (Stefek & Benes 1989). The antioxidant properties of stobadine were sug- gested to account for the mechanism of the cardioprotective and antihypoxic action of the compound (Benes & Stolc 1989).

This work was carried out to further investigate the effec- tiveness and the possible mechanism of the antioxidant ac- tion of stobadine using rat liver microsomes as a model system. Cumene hydroperoxide was chosen as the chemical agent because of its known ability to induce lipid peroxi- dation in a cytochrome P-450 mediated mechanism (Weiss & Estabrook 1986a & b). A preliminary account of this work was presented elsewhere (Stefek e t al. 1990).

Materials and Methods

Chemicals. Stobadine dihydrochloride, (-)-cis-2,8-dimethyl-2,3,4,- 4a,5,9b-hexahydro-lH-pyrido[4,3-b]indole dihydrochloride, was synthesized at the Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague (Benes & Stolc 1989). Sodium phenobarbital, sodium azide, carbon monoxide, thiobarbit- uric acid, N,N,N',N'-tetrahydro-methyl-p-phenylene diamine

(TMPD) and cumene hydroperoxide (80% in cumene) were pur- chased from Fluka AG (Buchs, Switzerland). Other chemicals were obtained from local commercial sources and were of analytical grade quality.

Preparation of microsomes. Male Wistar rats (220-270 g). fasted overnight, were used as liver donors. To induce enzyme activities the rats were pretreated with sodium phenobarbital for four days (3 x 80 mg/kg+ 1 x 40 mg/kg, intrapentoneally). Livers were hom- ogenized in 3 vol. of 1.15% KCI and centrifuged at 9,000 x g for 20 min. The supernatant was then centrifuged at 100,000 g for 60 min., the microsomal pellet collected and washed by resuspension in 3 vol. of 1.15% KC1 and recentrifugation. The washed pellet was stored under I mi of 1.15% KC1 at -80" for up to 2 weeks. For incubation the thawed pellet was resuspended in 1.15% KCl and diluted to the required concentration.

Incubations. For the assay of lipid peroxidation, the incubation mixture consisted of liver microsomes (1.2-1.7 mg protein/ml), cumene hydroperoxide (125 pM), MgCI, (6 mM) and 12.5 mM potassium phosphate buffer, pH 7.5. Incubation was started by addition of the microsomal suspension. Stobadine dihydrochloride was added dissolved in water. All incubations were conducted aerob- ically at 37" in a final volume of 1.0 mi.

Peroxidase activity of microsomal cytochrome P-450 was meas- ured at 25" using N,N,N',N-tetrahydromethyl-p-phenylene diamine (TMPD) as a hydrogen donor and cumene hydroperoxide as a substrate (O'Brien & Rahimtula 1978). The final reaction volume (2 ml) contained EDTA (1 .O mM), TMPD (0.2 mM), cumene hydro- peroxide (50-500 pM), microsomal protein (1.1 mg) and NaN, (0.1 mM) in 50 pM potassium phosphate buffer, pH 7.5. The rate of TMPD oxidation to Wurster's blue was determined spectrophoto- metrically at 610 nm during the first minute of the reaction and corrected for the rate of the reaction in the absence of cumene hydroperoxide.

Analytical methods. Protein concentration was determined accord- ing to the method of Geiger & Bessman (1972) and cytochrome P- 450 according to that of Omura & Sat0 (1964).

408 MILAN STEFEK ET AL.

A Fig. 1. Structure of stobadine, (-)-cis-2,8-dimethyl-2,3,4,4a,5,9b- hexahydro- 1 H-pyrido[4,3-b]indole.

The formation of thiobarbituric acid reactive products was meas- ured by the method of Buege & Aust (1978).

Cumene hydroperoxide level was assayed by a method of Hildeb- randt & Roots (1975) using ferrous ammonium sulphate and potas- sium thiocyanate.

Visible spectra were recorded on a Pye Unicam SP 1700 UV/VIS spectrophotometer. Absorbance readings for spectrophotometric assays were obtained using a Carl Zeiss VSU 2 UV/VIS spectropho- tometer.

Oxygen consumption was measured using a Clark-type oxygen electrode SOPS 31 (Chemoproject, Prague) according to Green & Hill (1984).

Results

Fig. 2 shows the effect of increasing concentrations of stoba- dine on cumene hydroperoxide-induced lipid peroxidation assessed by the accumulation of the thiobarbituric acid- reactive product in rat liver microsomes. Stobadine effec- tively inhibited thiobarbituric acid-reactive product forma- tion in a concentration dependent manner with the half- maximal inhibition value (ICs0) of 56 f 4.9 pM (n = 6); com- plete inhibition was achieved with stobadine concentrations above 1 mM. Yielding the same pattern, yet with IC,= 24 2.2 pM (n = 6), stobadine inhibited non-enzymatic lipid

A

eR W

5 1003

P a, Q 60

0 + & 20 li -

Stobadine (mM) Fig. 2. Effect of stobadine on thiobarbituric acid-reactive product formation in rat liver microsomes incubated with 125 pM cumene hydroperoxide. Control value for thiobarbituric acid-reactive prod- uct accumulation in the absence of stobadine: 14.1 nmol/mg protein/2.5 min. For experimental conditions see Materials & Methods. Each point represents the mean from at least three experi- ments.

peroxidation induced by ascorbate (0.5 mM)/ferrous iron (50 pM) in heat-denaturated microsomes (data not shown). Stobadine at the concentrations used in the above-men- tioned experiments did not interfere with the reaction be- tween thiobarbituric acid and malondialdehyde formed in the incubation mixture by decomposition of standard tetrae- thoxypropane (data not presented).

We examined the effect of stobadine on oxygen consump- tion by liver microsomes induced by cumene hydroperoxide. The addition of 125 pM cumene hydroperoxide to liver microsomes gives the maximum amount of oxygen uptake without the mixture becoming anaerobic (Weiss & Estab- rook 1986a). Oxygen uptake decreased with increasing con- centrations of stobadine (fig. 3). With increasing concen- trations of stobadine the initial rates of oxygen utilization and thiobarbituric acid-reactive product formation were in- hibited to the same degree as the extent of oxygen consumed and thiobarbituric acid-reactive product formed (data not shown).

Cumene hydroperoxide induced rapid and extensive de- struction of cytochrome P-450 characterized by more than 60% loss of the haemoprotein during 45-min. incubation in the presence of 125 pM hydroperoxide. In close agreement with the inhibition of thiobarbituric acid-reactive product accumulation and oxygen consumption, stobadine inhibited equally effectively the peroxidation-related degradation of cytochrome P-450, as shown in fig. 4.

Using the ferrous ammonium sulphate-potassium thyocy- anate method (Hildebrandt & Roots 1975) for measuring the concentration of hydroperoxides, we observed that the rate of cumene hydroperoxide consumption during the microsomal incubations was not affected by the presence of 0.1 or 1.0 mM stobadine (fig. 5).

Microsomal peroxidase activity was assayed using TMPD as a hydrogen donor and cumene hydroperoxide as a sub-

CumOOH

(PM) Buffer

1000

500

100

50

\\--- 25

L O

02=0

Fig. 3. Effect of stobadine on cumene hydroperoxide-dependent oxygen consumption in rat liver microsomal incubations. For ex- perimental condition see Material & Methods. Data from one repre- sentative experiment.

ANTIOXIDANT INHIBITION OF LIPID PEROXIDATION 409

r 201

U 4

U

Stobadine (mM) Fig. 4. Effect of stobadine on cumene hydroperoxide-induced de- struction of cytochrome P-450 in microsomal incubations. Incuba- tion time, 45 min. For other experimental conditions see Materials & Methods. Results are mean values from three experiments.

strate. Stobadine at concentrations ranging from 0.1 to 1.0 mM did not affect the rate of TMPD oxidation to Wurster's blue, established spectrophotometrically at 610 nm during the first minute of the reaction (data not shown).

Discussion

If reactive free radicals are produced in cells in ammounts sufficient to overcome the normally efficient protective mechanisms, metabolic and cellular disturbances can occur in various ways. A pathway dependent essentially on mem- brane damage can become fatal for a cell when uncontroled free radical induced peroxidation of polyunsaturated fatty acids occurs. At the subcellular level microsomes have been

n - 3

c W

r

E 0 0

3 0

0 2 4 6 a 10 04, t 7 7 I I , I , 1 - - 7 3 r I , I - I , , 3 I 9 8 . I I I I I I I I I 8 1 I - 1 1 I t

Time (min) Fig. 5. Time dependence of cumene hydroperoxide consumption in rat liver microsomal incubations. Effect of stobadine. No stobadine (0), 0.1 mM stobadine (A), 1 .O mM stobadine (0). For experimen- tal conditions see Materials & Methods. Each point represents the mean value from three experiments.

the subject of extensive investigation in the field of free radical initiated lipid peroxidation processes. The mem- branes of endoplasmic reticulum vesicles contain polyunsat- urated fatty acids in high proportions and therefore they are vulnerable to the peroxidative attack. At the same time they contain enzymes of the electron transfer systems which make them capable of producing highly reactive free radical species.

The results of our experiments clearly show that stobadi- ne effectively inhibited cumene hydroperoxide-dependent microsomal lipid peroxidation as evidenced by the decreased rate of the thiobarbituric acid-reactive product accumu- lation. Another line of evidence in support of the antioxi- dant action of stobadine is its inhibition of cumene hydro- peroxide-induced oxygen consumption in microsomal pre- parations. In this system the monitoring of oxygen consumption can be regarded as an index of lipid peroxi- dation since potential cumene hydroperoxide-driven and P- 450 mediated oxidation(s) of stobadine would not require molecular oxygen (Estabrook et al. 1975).

Potential metal chelating properties of stobadine do not appear to be important in its inhibitory action since cumene hydroperoxide-initiated lipid peroxidation is a process which neither requires iron nor is affected by its presence (Weiss & Estabrook 1986a). The absence of metal-chelating properties of stobadine was confirmed by the finding of Misik (1 991) that stobadine inhibited equally effectively DMPO trapping of hydroxyl radicals generated either by Fenton reaction or by the iron-independent photochemical decomposition of hydrogen peroxide.

With organic hydroperoxides, lipid peroxidation is in- itiated without the addition of electron donors such as NADPH and the typical primary initiation phase is omitted. The mechanism of cumene hydroperoxide-dependent lipid peroxidation involves homolytic cleavage of the -0-O- bond, catalyzed by cytochrome P-450, yielding the cumylox- yl radical which initiates lipid peroxidation by abstracting a hydrogen atom from the lipid (Weiss & Estabrook 198613; Thompson & Yumibe 1989). Since the presence of stobadine did not affect the rate of metabolic degradation of cumene hydroperoxide in microsomal incubations, the inhibition of cumene hydroperoxide bioactivation to reactive intermedi- ates does not seem to be the mechanism for the observed prevention of lipid peroxidation by stobadine.

Cytochrome P-450 is also considered an important endo- genous lipid peroxidation initiating species in the process called secondary initiation (Svingen et al. 1979; Bast & Haenen 1984; Ursini et al. 1989) in which primarily formed lipid hydroperoxides (LOOH) are cleaved into reactive oxy- gen radicals (LOO., LO., OH-). The same pattern of inhi- bition by stobadine of both cumene hydroperoxide-depend- ent enzymatic and irodascorbate induced non-enzymatic lipid peroxidation indicates that depression of cytochrome P-450 mediated secondary initiation is not obligatory for the inhibitory action of stobadine. This hypothesis is con- sistent with our observation that stobadine did not interfere with the peroxidase function of cytochrome P-450 moni-

410 MILAN STEFEK ET AL.

tored by determining the rate of cumene hydroperoxide- dependent oxidation of TMPD to Wurster’s blue. Inhibition of cytochrome P-450 reaction may imply inhibitor binding which can be monitored by spectral changes in the Soret absorption of cytochrome P-450. However, in the presence of stobadine in the concentrations ranging from 10 to 100 pM no spectral changes were observed in microsomes (data not shown).

Since stobadine did not prevent cumene hydroperoxide bioactivation, and since superoxide anion radicals and hy- droxyl radicals are not involved in cumene hydroperoxide- dependent lipid peroxidation (Weiss & Estabrook 1986b; Thompson & Yumibe 1989), the inhibitory effect of stobadi- ne may be at least partially explained by its scavenging of alkoxyl (LO.) and peroxyl (LOO.) radicals.

On the other hand, the interaction of peroxidized lipids with cytochrome P-450 is regarded as a possible cause for the destruction of the haemoprotein (Svingen et al. 1979; Poli et al. 1987). In our experiments we observed rapid destruction of microsomal cytochrome P-450 when cumene hydroperoxide was used to initiate peroxidation. As antici- pated from the inhibitory effects of stobadine on the peroxi- dation-related thiobarbituric acid-reactive product accumu- lation and on oxygen consumption, stobadine effectively prevented cumene hydroperoxide-dependent loss of cyto- chrome P-450.

The inhibitory effect of stobadine on lipid peroxidation does not seem to be unique for the microsomal membrane, since Ondrias et al. (1989) confirmed the effective antioxi- dant action of stobadine in pure phospholipid vesicles.

In conclusion, the above results obtained with the rat liver microsomal model system confirmed the antioxidant properties of stobadine and strongly indicate that it is pre- dominantly the ability of stobadine to scavenge alkoxyl and peroxyl radicals that is responsible for the observed antioxidant effects in cumene hydroperoxide treated micro- somes. Structurally related compounds, such as 3-methyl indole (Adams et al. 1987), 6-hydroxy- 1 ,4-dimethylcarbazo- le (Niki 1987; Malvy et al. 1980), indole-3-carbinol (Shertzer et al. 1987) and 5-hydroxy indole (Cadenas et al. 1989), were also shown to have free radical scavenging properties. Our results along. with the afore-mentioned findings re- ported for structural analogs of stobadine comply with the hypothesis of Adams et al. (1987) concerning the antioxi- dant properties of substituted indoles.

References

Adams, J. D., M. C. Heins & G. S. Yost: 3-Methyl-indole inhibits lipid peroxidation. Biochem. Biophys. Res. Commun. 1987, 149, 73-78.

Bast, A. & G. R. M. M. Haenen: Cytochrome P-450 and glutathio- ne: what is the significance of their interrelationship in lipid peroxidation. Trends Biochem. Sci. 1984, 9, 510-513.

Benes, L. & S. Stolc: Stobadine. Drugs Future 1989, 14, 135-137. Buege, J. A. & S. D. Aust: Microsomal lipid peroxidation. In:

Methods in enzymology, Vol. 52, Part C, Eds.: S. Fleischen and L. Packer. Academic Press, 1978, pp. 302-310.

Cadenas, E., M. G . Simic & H. Sies: Antioxidant activity of 5- hydroxytryptophan, 5-hydroxyindole, and DOPA against microsomal lipid peroxidation and its dependence on vitamin E. Free Rad. Res. Comms. 1989, 6, 11-17.

Estabrook, R. W., J. Werringloer, E. G. Hrycay, P. J. OBrien, A. D. Rahimtula & J. A. Peterson: Cytochrome P-450 an oxygenase, peroxidase and oxidase. Biochem. SOC. Duns. 1975, 3, 811-813.

Geiger, P. J. & S. P. Bessman: Protein determination by Lowry’s method in the presence of sulfhydryl reagents. Andy?. Biochem. 1972,49,467473.

Green, M. J. & H. A. 0. Hill: Chemistry of dioxygen. In: Methods in enzymology, Vol. 105. Ed.: L.Packer. Academic Press 1984, pp. 3-22.

Hildebrandt, A. G. & I. Roots: Reduced nicotine adenine dinucleot- ide phosphate (NADPH)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes. Arch. Biochem. Biophys. 1975,171,385-397.

Horakova, L., L. Lukovic & S. Stolc: Effect of stobadine and vitamin E on the ischemic reperfused brain tissue. Pharmazie

Malvy, C., C. Paoletti, A. J. F. Searle & R. L. Willson: Lipid peroxidation in liver: hydroxydimethylcarbazole, a new potent inhibitor. Biochem. Biophys. Res. Commun. 1980, 95, 734737.

Misik, V.: Effects of antioxidants on membrane lipid peroxidation. Ph.D. Theses, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava 1991.

Niki, E.: Antioxidants in relation to lipid peroxidation. Chem. Phys. Lipids 1987,44, 227-253.

O’Brien, P. J. & A. D. Rahimtula: A peroxidase assay for cyto- chrome P-450. In: Method in enzymology, Vol. 52, Part C. Eds.: S. Fleischen and L. Packer. Academic Press 1978, pp. 407412.

Omura, T. & R. Sato: The carbon monoxide-binding pigment of liver microsomes. J. Biol. Chem. 1964, 239, 2370-2378.

Ondrias, K., V. Misik, D. Gergel & A. Stasko: Lipid peroxidation of phosphatidylcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic- antihypoxic drug stobadine. Biochim. Biophys. Acta 1989, 1003,

Poli, G., E. Albano & M. U. Dianzani: The role of lipid peroxi- dation in liver damage. Chem. Phys. Lipids 1987,45, 117-142.

Shertzer, H. G., M. P. Niemi, F. A. Reitman, M. L. Berger, B. L. Myers & M. W. Tabor: Protection against carbon tetrachloride hepatotoxicity by pretreatment with indole-3-carbinol. Exp. Mol. Parhol. 1987, 46, 180-189.

Stasko, A., K. Ondrias, V. Misik, H. Szocsova & D. Gergel: Stobadi- ne - a novel scavenger of free radicals. Chem. Papers 1990, 44,

Stefek, M. &. L. Benes: Hydrogen peroxide-dependent liver microsomal N-demethylation and N-oxygenation of stobadine, a gamma-carboline antiarrhythmic and cardioprotective agent. Xenobiotica 1989, 19, 627-634.

Stefek, M., M. Zimanova & L. Benes: Stobadine inhibition of microsomal lipid peroxidation. Proceedings ofthe VZZZrh Znterna- tional Symposium on Microsomes and Drug Oxidations Stockholm, Karolinska Institutet, 1990, June 25-29, p. 155.

Stolc, S., V. Bauer, L. Benes & M. Tichy: Stobadine - das Heilmittel besonders mit antiarrhythmischer und subsidiarer Wirkung bei der Hypoxie des Myokard, sowie ein Verfaren zu ihrer Herstel- lung. Swiss Patent 1985, 651-754.

Stolc, S. & L. Horakova: Effect of stobadine on postischemic lipid peroxidation in the rat brain. In: New trends in clinical neurophar- macology. Eds.: D. Bartko et al. John Libbey, London 1988, pp.

Svingen, B. A,, J. A. Aust, F. O’Neal & S. D. Aust: The mechanism of NADPH-dependent lipid peroxidation. The propagation of lipid peroxidation. J. Biol. Chem. 1979, 254, 5892-5899.

Thompson, J. A. & N. P. Yumibe: Mechanistic aspects of cyto- chrome P-450-hydroperoxide interactions: substituent effects on degradative pathways. Drug Metub. Rev. 1989, 20, 365-378.

1990, 45, 223-224.

238-245.

493-500.

59-63.

ANTIOXIDANT INHIBITION OF LIPID PEROXIDATION 41 1

Weiss, R. H. & R. W. Estabrook: The mechanism of cumene hydro- peroxide-dependent lipid peroxidation: the function of cyto- chrome P-450. Arch. Biochem. Biophys. 1986b, 251, 348-360.

Williams, C. H. & H. Kamin: Microsomal triphosphopyridine nu- cleotide-cytochrome c reductase of liver. J. Biol. Chem. 1962,237,

Ursini, F., M. Maiorino, P. Hochstein & L. Ernster: Microsomal lipid peroxidation: mechanism of initiation. The role of iron and iron chelators. Free Radical Biol. Med. 1989, 6, 31-36.

Weiss, R. H. & R. W. Estabrook: The mechanism of cumene hydro- peroxide-dependent lipid peroxidation: the significance of oxygen uptake. Arch. Biochem. Biophys. 1986a, 251, 336-347. 58 7-59 5 .