Effects of phenols on eicosanoid synthesis in A23187-stimulated human whole blood

Prostaglandins

EFFECTS

45: 193-20 1, 1993

OF PHENOLS ON EICOSANOID SYNTHESIS STIMULATED HUMAN WHOLE BLOOD J.

Department of Biomedical P.O. Box 607,

IN A23187-

Alanko

Sciences, University of SF-33101 Tampere, Finland

Tampere,

ABSTRACT We have previously demonstrated that catecholamines have opposite effects on leukotriene (LT) and prostanoid synthesis. The aim of the present study was to test the effects of phenols (catechol, hydroquinone, phenol and resorcinol) on LTB LTE and thromboxane (TX)B synthesis in A23187-stimulated human who4’e bloolf. All tested compounds inhi?i ited LTB4 and LTE synthesis. The IC values for catechol were 3 PM, 6 PM; for hydroqumone 4 p M, 3 FM; for phenol % 5 PM, 226 PM and for resorcinol 180 PM, 902 PM. The compounds did not stimulate TXB2 synthesis but only inhibited it. The IC value for catechol was 3 PM, for hydroquinone 7 PM, for phenol 18 FM and Yor resorcinol 25 PM. Catechol and hydroquinone had hardly any effect on the LT/TX ratio. Phenol and resorcinol even increased the LT/TX ratio. The positions of hydroxyl groups of phenolic compounds are thus important for their actions on the LT/TX ratio. INTRODUCTION Phenolic compounds have a dual effect on prostaglandin H synthase (PGHS): at low concentrations they stimulate (1,2) and at high concentrations they inhibit it (3). They stimulate PGHS acting as cosubstrates for the peroxidase component of PGHS (4-6) and/or defending the PGHS against self-inactivation (7,8). Phenolic compounds can also inhibit prostanoid synthesis directly (9) and/or by reducing the hydroperoxide tone to a level at which PGHS cannot be activated (10). On the other hand, many inhibitors of leukotriene synthesis have a phenolic structure, these include nordihydroguaiaretic acid (11,12) and caffeic acid (13). Most of the information available on the effects of phenolic compounds on arachidonic acid cascade is based on work with enzyme preparations (12,14). These provide useful models for studying the mechanism in detail, but they do not help to identify the effects of phenolic compounds on 5lipoxygenase and PGHS pathways of arachidonic acid at the same time and in cellular environments. We have previously demonstrated that catecholamines, which also have a phenolic structure, have opposite effects on the formation of leukotriene (LT)B4 and prostaglandin (PG)E;! in A23187-stimulated human polymorphonuclear leukocytes (PMNs) (15). The same effect was also seen when A23187-stimulated whole blood was used as a model, and LTB4 and thromboxane (TX)B2 synthesis was measured (16). Phenols (catechol, hydroquinone and phenol) also had opposite effects on LTB4 and PGE$ synthesis in A23187-stimulated PMNs (17), although the stimulatory effect on PG synthesis was weaker than that of catecholamines (15). Laughton et al. have also“Lh s own that hydroquinone inhibits LTB synthesis in rat peritoneal leukocytes, however hydroquinone did not modulate TX %2 synthesis in rat leukocytes (18). Stimulated whole blood is regarded as one of the best models to study leukotriene synthesis in vitro (19). This model takes into account the complex interactions between the different cell types capable modulating eicosanoid synthesis and metabolism (20-24). Some inhibitors of the synthesis of LTB4 are active only in

Copyright 0 1993 Butterworth-Heinemann

194

Prostaglandins

PMNs but not in whole blood (25), where drug binding to proteins or cell membranes may operate. The purpose of the present study was to shed further light on our earlier finding and examine the effects of phenols (catechol, hydroquinone, phenol and resorcinol) on LT/TX ratio in A23187-stimulated whole blood. LTB4 and LTE4 were measured to monitor both pathways of LTA4 metabolism. TXB2 was used as an indicator of cyclooxygenase activity. It is a new point of view, that phenolic compounds could modulate the balance between 5lipoxygenase and PGHS pathways of arachidonic acid (for a recent review, see 26). Pure enzymes are better models to study the mechanism of phenols to modulate arachidonic acid metabolism in detail, Hsuanyu and Dunford (27) published recently an extensive investigation on the mechanism of phenols to modulate PGHS. However, investigations with more complicated models are also needed. If phenols are active in such a complex model as in A23187stimulated whole blood, they should also have some physiological importance in vivo. MATERIALS AND METHODS Chemicals. Ca ionophore A23187, catechol, hydroquinone and resorcinol were from Sigma Chemicals Co. (St Louis, MO, USA) Phenol was purchased from E. Merck (Darmstadt, Germany). LTB4, LTE4, 3H-LTC4 and 3H-TXB were purchased from Amersham International (Buckinghamshire, England). L&4 was obtained from Cayman Chemical (Ann Arbor, MI, USA) and unlabelled TXB2 was from Upjohn Diagnostics (Kalamazoo, MI, USA). Whole blood incubation. Freshly drawn heparinized venous blood was obtained from healthy volunteers who had not taken any drugs for at least two weeks. The final concentration range was 0.18 PM to 1.8 mM for catechol, hydroquinone, phenol and resorcinol, they were added in 0.9% NaCl. Eicosanoid synthesis of whole blood was triggered by Ca ionophore A23187 (final concentration 10 PM), the incubation was carried out for 60 min at 37°C. Plasma was separated by centrifugation at +4”C 1600 g for 10 min; and stored at -20°C until further processing (16). Q determination. LTB4 was extracted by using C8 solid-phase cartridges and LTB4 was separated and quantitated by RP-HPLC as described earlier by us (16). 9. determination. Cysteinyl leukotriene formation was determinated as LTE4- Ike immunoreactivity from unextracted plasma by RIA. An in-house raised against the bovine serum albumin conjugate of rabbit antiserum, LTC4/LTD4/LTE , was used for the assay. The cross reactivity values of the antibody used m L4r E RIA were: LTC4 100 %, LTD4.90 %, LTE 35 %, LTF4 120 %, S(S),6(R)-diH&TE 7.33 % and to other eicosanoids less th the LTE was determined in a cross reactive way, using gs%‘as radiolabe 4 ed ligand and LTE4 as the non-labeled ligand. In ten independe!t assays, the detection limit and I&o value obtained were 4.38 + 2.1 and 43.0 + 9.6 pg/tube, respectively (28). >” The samples were diluted 1:150-300 in assay buffer. The compounds included in the study *were checked for cross-reaction at the highest concentration employed, and no interference was found. TXB;? determination. The samples were diluted 1:lOOO in assay buffer and TXB was measured using a direct RIA with double antibody separation as descn2b ed earlier (29). Antiserum was received from Prof. C. Taube (Martin Luther University, Halle, Germany). The compounds included in the study were checked for cross-reaction at the highest concentration employed, and no interference was found.

Prostaglandins

195

RESULTS The control synthesis (A23187-stimulation only) of LTB4 was 42 + 4 ng/ml (mean &- SEM, n=16), that of LTE 36 + 6 ng/ml (n=l6), and that of TXB 167 ..$. . + 26 ng/ml (n=16). All phenols m lb&d LTB4 and LTE4 synthesis (Fig. T and 2). Catechol, hydroquinone and phenol had similar inhibitory effects on LTB4 and LTE4 synthesis. Resorcinol had a lesser inhibitory effect on LTE4 synthesis than on LTB4. Catechol and hydroquinone, whose dose response curves are almost identical, are about 100 times more potent inhibitors of leukotriene synthesis than phenol and resorcinol. All the phenols inhibited TXB2 synthesis and no stimulation was seen. Catechol and hydroquinone were the most potent inhibitors of TXB synthesis (Fig. 3); they are about equipotent inhibitors of both thromboxane an 2 leukotriene synthesis. The concentrations at which phenol and resorcinol inhibited TXB2 synthesis were about 100 times lower than those needed to inhibit leukotriene synthesis. However, catechol and hydroquinone are 3-10 times more potent inhibitors of TXB synthesis than phenol and resorcinol. The IC values at which phenols inhibited 2 TB4, LTE4 and TXB2 synthesis, are shown in‘?able 1.

0.18

1.8

18

Fig 1. Effects of catechol (m), hydroquinone LTB4 synthesis in A23187-stimulated

180

1800

@Ml

(IJ), phenol (o), and resorcinol (0) on

human whole blood (mean + SEM, n = 4).

196

Prostaglandins

Table 1. IC50 values at which phenols inhibited LTB4, LTE4 and TXB2 synthesis, calculated from the dose response curves. LTB4

LTE4

TXB2

Catechol

3pM

6crM

3crM

Hydroquinone

4CLM

3pM

7/4M

Phenol

285 /AM

226 /AM

18 /.LM

Resorcinol

180 FM

902 /.LM

25 /LM

25

0.18

1.8

18

Fig 2. Effects of catechol (=), hydroquinone LTE4 synthesis in A23187-stimulated

180

1800

(Cl), phenol (o), and resorcinol (0) on

human whole blood (mean + SEM, n = 4).

197

Prostaglandins

0 0.18

1.8

18

Fig 3. Effects of catechol (m), hydroquinone TXB2 synthesis in A23187-stimulated

180

1800 CUM)

(Et), phenol (o), and resorcinol (0) on

human whole blood (mean +. SEM, n = 4).

DISCUSSION A large number of known lipoxygenase inhibitors such as phenols can be classified as antioxidants or radical scavenging agents. A possible key to the inhibition of leukotriene synthesis is that phenols inhibit !I-lipoxygenase by reducing the catalytically active Fe(II1) enzyme to the catalytically inactive Fe(I1) form (12,30). When using whole cell system, it is not possible to measure the the oxidation state of the enzyme iron. However, the reducing potentials of phenols might be used as their indicators to reduce the enzyme Fe(lI1) (31). Whole cell system should be used to investigate the effects of phenols on leukotriene synthesis, although the mechanism cannot be studied as detailed as when using pure enzyme. However, whole blood incubation, where cell to cell interactions and protein binding may operate, is closer to physiolocigal conditions than pure enzyme. The present findings on the effects of phenols on the synthesis of LTB4 in whole blood are similar to those reported recently in PMNs by us (17). With the exception of resorcinol, the compounds tested had almost the same inhibitory effect on cysteinyl leukotriene synthesis measured as LTE4-like immunoreactivity as on LTB synthesis. synthesis at lower concentrations than LTE I?esorcinol inhibited LTB synthesis. One possible exp4anabon . IS that resorcinol modulates cysteiny 4 leukotriene breakdown and so affects LTE4-like immunoreactivity assay. On this basis it may be concluded that catechol, hydroquinone and phenol - and possibly

198

Prostaglandins

resorcinol - inhibit leukotriene synthesis by affecting the 5lipoxygenase enzyme rather than the speckfit synthases of LTB4 or cysteinyl leukotrienes. All the uhenols tested were inhibitors of leukotriene synthesis in whole blood, and active even in lower concentrations than in PMNs as we previously reported (17). On the other hand, the maximal inhibition of leukotriene synthesis by the phenols and caffeic acid (a catecholic compound without adrenergic activity) (15) in whole blood was higher than that of catecholamines (15), although catechol, hydroquinone, caffeic acid and catecholamines were about equipotent inhibitors of LTB4 synthesis in PMNs (15,17). This might be explained by the fact that the phenols and caffeic acid are more stable than catecholamines in whole blood incubation. Phenol (monohydroxylbenzene) and resorcinol (meta-dihydroxylbenzene) are poor inhibitors of leukotriene svnthesis. This means. that the ontimal oositions of the hydroxyl =roups of the benzene ring for the inhibition of lebkotriene synthesis are the ortho- (catechol) and para-positions (hydroquinone). Since phenols also act to inhibit leukotriene synthesis in whole blood, it may be assumed that this action is also visible in the presence of plasma proteins and complex interactions between different cell types. It seems that the inhibition of leukotriene synthesis could be a chemical interaction between the phenols and 5-lipoxygenase. A key to the inhibition of leukotriene synthesis is that phenols inhibit 5-lipoxygenase by reducing the catalytically active ferric enzyme to the catalytically inactive ferrous form (12,30,31). The inhibitory potency of phenols correlates very well with their reductton potentials (V), which are 0.53 for catechol, 0.46 for hydroquinone, > 0.80 for phenol and 0.81 for resorcinol at pH 7 (32). In the present study the reduction potential of phenols seems to indicate their capability to inhibit the 5lipoxygenase, although the redox properties are not the only thing, that modulates the inhibitory potencies of 5-lipoxygenase inhibitors as shown with pure soybean lipoxygenase (31). However, hydrophobicity does not further explain the different potencies of various phenols to inhibit 5-lipoxygenase in the present study. It is well known, that phenols at low concentrations stimulate PGHS, but at high concentrations they inhibit PGHS (27). However, most of the available information on the effects of phenols on PGHS is based on work with enzyme preparations (2-4,9), and antioxidants such as phenol and hydroquinone have often been included in the reaction mixture. Their influence must therefore be taken into consideration, when evaluating the data (9,lO). To investigate the physiological relevance of the studies with pure enzymes and isolated cells, complicated models such as stimulated whole blood should be used. The effects of phenols on TXB synthesis in A23187-stimulated whole blood were different from their effects on ItiGE+ in A23187-stimulated PMNs reported by us (17). They all inhibited TXB2 syntheses in whole blood, and no stimulation was seen. Although all phenols except resorcinol stimulated PG synthesis in PMNs at the low concentrations tested, they also inhibited PGH&2 synthesis at high concentrations, and in the latter resorcinol was the most potent inhibitor (17). That phenols do not stimulate TXB2 synthesis is supported by the results of Laughton et al. (18). When using rat peritoneal leukocytes they found, that hydroquinone does not modulate TXB synthesis, although it inhibits LTB4. synthesis. This is further supported by the ;L mdings of Koshihara et al. that caffetc acid does not stimulate only inhibit TXB synthesis in isolated platelets (13). However, caffeic acid is able to stimulate the l& HS of rat mast cells (13). Thus, the results seem to be different, if PGE2 or TXB2 is used as an indicator of PGHS activity. However, catecholamines stimulated TXB synthesis in A23187-stimulated whole blood (16) as they did also in PMNs (13. Why catecholamines are able to stimulate TXB2, synthesis in whole blood, but phenols are not able to stimulate TXB2 synthesis in whole blood, although they stimulated PGFC2 synthesis in PMNs,

Prostaglandins

remains open. In the future other prostanoids than thromboxane should also be measured, because phenols might modulate the PGHS product profile (1). Catecholamines and caffeic acid were more potent stimulators of PGE2 synthesis in PMNs (15) than any of the phenols (17). Catecholamines also stimulated TXB2 synthesis in whole blood. However, caffeic acid had no effect on TXB synthesis m whole blood (16), nor did it stimulate TXB2 synthesis in isolated plate l2ets but only inhibited it at high concentrations (13). The side chain of catecholamines and caffeic acid seem to play very important role in affecting PGHS and especially thromboxane synthesis. The me&positions of the hydroxyl groups of the benzene ring were thus optimal for the inhibition of PG synthesis in PMNs. In whole blood the ortho- and para-positions are optimal for % t e inhibition of TXB synthesis. This means that the role of various prostanoids in monitoring the effect o$ drugs on PGHS activity may vary depending on the model used. The effect of phenols on pure thromboxane synthase should be investigated, because there exists no data, if phenols might modulate thromboxane synthase activity. The difference between phenols and catecholamines (16) on TXB synthesis in A23187-stimulated whole blood is an important finding. In pure PGH s both phenols and catecholamines have stimulatory action at low concentrations (1,27) and the same kind of effect is also seen, when PMNs are used as a model and PGE2 measured (15,17). To find out the reason, why i) hydroquinone stimulates PGE2 but not TXB2 synthesis in PMNs and ii) catecholamines are able to stimulate TXB2 synthesis in whole blood but phenols are not, would increase our knowledge of the physiological regulation of PGHS. Catecholamines and caffeic acid decreased the leukotriene/prostanoid ratio in PMNs (15) and in whole blood (16). The effects of phenols on the LT/PG ratio is different. In PMNs catechol, hydroquinone and phenol decreased the LTB,/PGE2 ratio, while resorcinol increased it (17). In whole blood, catechol and hydroquinone did not modulate the LT/TX ratio because they inhibited both leukotriene and thromboxane synthesis equipotentially. However, phenol and resorcinol increased the LT/TX ratio in whole blood. Although compounds with similar structures have more or less similar effects on each eicosanoid studied, their effects on the ratio between various eicosanoids can be totally different. The structure of the phenolic compound, the positions of hydroxyl groups and side chain, are thus critical factors with regard to its effects on the leukotriene/prostanoid ratio. This should be taken into consideration in developing phenolic compounds for drugs that affect on eicosanoid synthesis. Physiological compounds with a phenolic structure and sufficiently high tissue concentrations might modulate eicosanoid synthesis, especially leukotriene/prostanoid ratio (for a recent review, see 26). ACKNOWLEDGEMENT This work was supported by Astra in Finland, the Emil Aaltonen Foundation, the Finnish Cultural Foundation (Pirkanmaa), the Finnish Medical Foundation, Hoechst Fennica, the Jalmari and Rauha Ahokas Foundation and the Pharmacal Research Foundation.

200

Prostaglandins

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Editor:

W.E.M.

Lands

Received:

8-12-92

Accepted:

12-30-92