Effects of Oxygen Radicals on Lipoxygenases 819

Effects of Oxygen Radicals, Hydrogen Peroxide and Water-soluble Singlet Oxygen Carriers on 5- and 12-Lipoxygenase Klaus Muller* and Klaus Ziereis

Institute of Pharmacy, University of Regensburg, P.O. Box 101042, D-8400 Regensburg, Germany

Received October 14, 1992

Wirkung von Sauerstoffradikalen, Wasserstoffperoxid und wasserlos- lichen Singulettsauerstoff-Tragern auf 5- und 12-Lipoxygenase

Die Hemmung der 5-Lipoxygenase von polymorphkernigen Rinder-Leu- kocyten und der 12-Lipoxygenase von Rinder-Thrombocyten durch aktive Sauerstoffspezies wurde untersucht. Sauerstoffradikale und Wasserstoff- peroxid zeigten eine deutliche Hemmung der 12-Lipoxygenase, wiihrend sie die 5-Lipoxygenase-Aktivitat nur in geringem Ausmd beeintrachtig- ten. Aus wasserloslichen Naphthalin-Endoperoxiden freigesetzter Singulett Sauerstoff war ohne Effekt.

Inhibition of 5-lipoxygenase from bovine polymorphonuclear leukocytes and 12-lipoxygenase from bovine platelets by active oxygen species has been studied. Oxygen radicals and hydrogen peroxide markedly inhibited 12-lipoxygenase, whereas 5-lipoxygenase activity was only moderately influenced. Singlet oxygen liberated from water-soluble naphthalene endo- peroxides was without effect.

The products of 5-lipoxygenase (5-L0), particularly leukotriene B4 (LTB4), and 12-hydroxyeicosatetraenoic acid (1 2-HETE) of the 12-LO pathway have been implicated in the development of inflammatory skin diseases, such as psoriasis’). The etiology of psoriasis is still unknown’), but a characteristic of lesional skin is the elevated levels of these oxygena- tion products of arachidonic acid.

The oxygenation reactions of LO involve free radical intermediates’) and even singlet oxygen4). Moreover, the generation of hydroxyl or an equivalent oxygen radical by conversion of various hydroper- oxyeicosatetraenoic acids (HPETEs) to HETEs has been suggested5). Accordingly, many LO inhibitors may be classified as antioxidants or radical scavengers, whereas on the other hand 5-LO is prone to inactivation by peroxides and even a possible inactivation by superoxide (‘02-) and hydroxyl radicals (’OH) has been proposed6). Consequently, active oxygen species may play an ambiguous role in arachidonic acid metabolism. They may function as activators of LO as well as suppressors of eicosanoid biosynthesis in a self-catalyzed inactivation and may be able to serve as “suicide” substrates’). These observations are in line with the generation and/or scavenging of oxygen radicals or singlet oxygen by antipsoriatic drugss). For instance, benoxaprofen, which is a singlet oxygen (lo2) sensitizer9), inhibits the LO and is beneficial in psoriasislO). Likewise anthralin (dithranol), an effective antipsoriatic agent, has been shown to inhibit both 5-LO”) and 12-LO”). In previous studies we have reported on the formation of ‘0, and oxygen radicals by anthralin’3*’4z’5). So there might be a correlation between LO inhibition and the generation of active oxygen species. The direct effect of active oxygen species on 5- and 12- LO activity has not yet been established.

In this study, we have examined the effects of active oxygen species on the LTB, or 5-HETE production by bovine polymorphonuclear leukocytes (PMNL) and 12- HETE production by bovine platelets.

Materials and Methods

Chemicals

Arachidonic acid, calcium ionophore A23 187, chelating resin (sodium form), diethylenetriaminepentaacetic acid (DTPA), histopaquea-1077,

nordihydroguaiaretic acid, xanthine, xanthine oxidase (EC 1.1.3.22) were from Sigma; Munich, Germany; 5-HETE, I2-HETE, 5S,12S-diHETE, 12- HHT, LTB4, PGB’ (Paesel GmbH FrankfurtM., Germany); FeC13 6H20, FeS04 . H20, hydrogen peroxide (30%), nitro blue tetrazolium (NBT) were obtained from Merck; Darmstadt, Germany; solvents for HPLC were of HPLC quality (Roth; Karlsruhe, Germany); bovine blood was obtained from the local slaughterhouse.

Active oxygen species generating systems

In order to produce a flux of ‘Oy a system of xanthine oxidase (XO, 0.02 U/mL) and xanthine (30 pM) was used’@. Generation of ’02- was ascertained spectrophotometrically by monitoring the reduction of NBT at 560 nrn. XO was added last to initiate the reaction. The phosphate buffer solution was passed through a column of chelating resin to remove trace levels of iron. DTPA (0.1 mM) was added to prevent hydroxyl radical formation.

Hydroxyl radical was generated by a superoxide driven Fenton reaction”) with the xanthine/XO system described above by addition of 0.1 mM FeS04 . HzO and 0.1 mM DTPA. Controls were performed with the ferrous salt, H202, xanthine, and the chelators each alone. Additionally, hydroxyl radicals were produced in a Fenton reaction with hydrogen peroxide (17.6 pM) and Fe2+-DTPA (0.1 mM). H202 was added last in three portions to initiate the reaction.

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Figure 1

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Miiller and Ziereis

Singlet oxygen was produced by thermal decomposition of water- soluble endoperoxides of naphthalene derivatives (30 pM, Fig. 1) with dif- ferent partition coefficients and half-lives between 25 and 52 min, as described‘”’.

5-Lipmygenase assay

PMNL were prepared essentially as described”) from Na-EDTA- anticoagulated bovine blood. Contaminating platelets were removed by repeated centrifugations at 100 g for 20 min. The purified PMNL were suspended at a concentration of 1 x lo7 cells/mL in phosphate buffered saline (PBS) (composed of 8.00 g NaCl, 0.20 g KCI, 1.00 g Na,HPO, 2H,O, 0.15 g NaH2P0, H,O, 0.20 g KH,PO,, adjusted to pH 7.4 with 3 N NH, in a final volume of 1000 mL bidist. H20). Cells were counted with a Sysmex microcellcounter CC-130 attached to an auto dilutor AD-241.

Preincubation was performed with 2.4 mL of the suspension and 10 pL of active oxygen species generating systems at the desired concentrations in PBS (endoperoxides in DMSO) or vehicle control (DMSO at final concentration of 0.4%) for 30 rnin at 37°C in a shaking water bath. The syntheses of LTB4 and 5-HETE were stimulated by the addition of CaC1, and calcium ionophore A23187 (final concentrations 2 mM and 20 pM). The incubation was allowed to proceed for 10 min at 37°C. Then the incubation was terminated by the addition of 3.0 mL MeOH/CH3CN (1+1) containing NDGA as a scavenger of active oxygen species (final concentration 0.01 mM) and prostaglandin B2 as an internal standard (final concentration 0.3 pM) and the incubation mixture was kept in an ice bath for 20 min. After centrifugation at 4000 g for 5 min at 4°C the supernatant was diluted with 5 mL of water and applied to a prewashed octadecylsilane reversed phase cartridge (Baker), which had been washed with 10 mL of MeOH and 5 mL of water. The eicosanoids were eluted with 3 mL of MeOH, diluted with 3 mL of water and subjected to reverse phase HPLC analysis.

HPLC was performed on a 250 x 4 mm column packed with Nucleosil Ci8 (7 pm particles; Bischoff, Leonberg, Germany). The isocratic elution conditions of LTB, were THF/MeOH/water (25+30+45, vol/vol), plus 0.1 vol % acetic acid, adjusted to pH 5.5 with 3N NH3, at a flow rate of 0.9 mL/min (Kontron 420 pump), monitored at 280 nm with a Kontron 735 LC UV detector, whereas 5-HETE was monitored at 232 nm using MeOH/water (77+33, vol/vol), plus 0.1 vol % acetic acid, pH 5.5, flow rate 0.9 mL/min. Integrated areas of the peaks were compared to the PGB, internal standard and to external standards of authentic samples. Molar absorption coefficients of Samuelsson et al.”) were used for calculations. % Inhibition of the formation of LTB4 or 5-HETE by bovine PMNL was calculated by the comparison of active oxygen generating system (N = 3, SD < 10%) with control activity (N = 8, SD < 5%).

12-Lipoxygenase assay

The procedure was similar to the one described for 5-LO with the following modification for cell preparation: The platelets were prepared from Na-EDTA-anticoagulated bovine blood. After centrifugation at 100 g for 20 min the platelet-rich plasma was removed by aspiration. The platelets were collected by centrifugation at 1000 g for 15 min and the platelet pellet was suspended in PBS at a volume equal to one third of the original plasma volume. The suspension was centrifuged at 1000 g for 15 min and the washed platelets were resuspended at a concentration of 1 x lo7 cells/mL in PBS. 12-HETE was detected at 232 nm.

Results and Discussion

Inhibition of 5- and 12-LO by active oxygen species has been investigated with PMNL and platelets from bovine blood, because it can be easily obtained in large quantities and the production of LTB, by bovine PMNL’9,21) and 12- HETE by bovine platelets22) has been well established. Table 1 shows the influence of various active oxygen species producing systems on the formation of 5-LO products from bovine PMNL and 12-LO products from bovine platelets.

Table 1 shows that 12-LO is markedly susceptible to oxygen radicals and H202, whereas 5-LO is only moder- ately inhibited by these active oxygen species. On the other hand, ‘02 generated by the thermal decomposition of naphthalene endoperoxides was completely ineffective. A recent study showed that LO inhibition by phenidone and BW755C only occurs after oxidative activation of the drugs by the peroxidase-like activity, and reactive species formed during this process, among others ‘ 0 ~ and H202, have been suggested for the ina~tivation~~).

The results from our studies indicate that in the case of 12-LO oxygen radicals and H202 are capable of enzyme inactivation at concentrations that may be produced by drugs acting as inhibitors of LO. Therefore, a mechanism for enzyme inactivation which involves the participation of active oxygen species appears likely for certain 12-LO inhibitors, but not for 5-LO inhibitors, because 5-LO activity is only slightly affected by active oxygen species. In addition, the production of ‘02 by LO inhibitors does not contribute to their mechanism of inactivation of both en- zymes.

Table 1: Inhibition of 5-LO and 12-LO by active oxygen species

5-LO 12-LO (PMNL) (Platelets)

% Inhibition % Inhibidon Active oxygen species generating system

’02- xanthine (30 )IM)/XO (0.02 U/mL) 15 89

‘OH FeSOA-DTPA (0.1 mMYxanthine (30 )IM)/XO (0.02 U/mL) 15 87

FeS04-D-A (0.1 mM)/H~O~ (17.6 PM) 17 85

H2Oz HzOz (17.6 W) 17 88

‘0, naphthalene endoperoxides (30 NM, Fig. 1)

Inhibition was calculated by the comparison of the mean values of test system (n = 3) with control (n = 6-8), range < 10%. Controls with FeSO,, DTPA or xanthine each alone did not influence 5-LO and 12-LO activity.

Arch. Pharm. (Weinheim) 326,819-821 (1993)

Effects of Oxygen Radicals on Lipoxygenases

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Arch. Pharm. (Weinheim) 326,819-821 (1993)