PROSTAOINS LEXJKOTRIENES AM)EssEMTIAL FAWACIDS

Effect of tert-Butyl Hydroperoxide on Cyclooxygenase and Lipoxygenase Metabolism of Arachidonic Acid in Rabbit Platelets Y. Fujimoto. Department

S. Takai, K. Matsuno, T. Sumiya, H. Nishida, S. Sakuma and T. Fujita of Hygienic Chemistry,

Osaka University of Phar-mace~~ticcrl Scierlces. Matsuhal-a, Osaka 580. Japarl

(Reprint requests to YFI

The effect of tert-butyl hydroperoxide (t-BOOH) on the formation of thromboxane (TX) B,, 12hydroxy-5, 8, lo-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid (AA) in washed rabbit platelets was examined. t-BOOH enhanced TXB, and HHT formation at concentrations of 8 PM and below, and at 50 PM it inhibited the formation, suggesting that platelet cyclooxygenase activity can be enhanced or inhibited by t-BOOH depending on the concentration. tBOOH inhibited 1tHETE production in a dose-dependent manner. When the platelets were incubated with 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) instead of AA, t-BOOH failed to inhibit the conversion of 1ZHPETE to 12-HETE, indicating that the inhibition of 1ZHETE formation by t-BOOH occurs at the lipoxygenase step. Studies utilizing indomethacin (a selective cyclooxygenase inhibitor) and desferrioxamine (an iron-chelating agent) revealed that the inhibitory effect of t-BOOH on the lipoxygenase is not mediated through the activation of the cyclooxygenase and that this effect of t-BOOH is due to the hydroperoxy moiety. These results suggest that hydroperoxides play an important role in the control of platelet cyclooxygenase and lipoxygenase activities.

ABSTRACT.

INTRODUCTION In many tissues and cells, two distinct enzymic pathways exist for the oxygenation of arachidonic acid (AA). Prostaglandins (PGs), prostacyclin and thromboxanes (TXs) are formed by the cyclooxygenase pathways. whereas the various polyunsatured hydroxy fatty acids are formed by the lipoxygenase pathways. In washed platelets, AA is converted into TXAz and 12-hydroxy-5. 8, lo-heptadecatrienoic acid (HHT) by the cyclooxygenase pathway and into 12-hydroxy-5,8,10, lil-eicosatetraenoic acid ( I2-HETE) by the lipoxygenase pathway (1). TXAz is a potent vasoconstrictor and inducer of platelet aggregation and rapidly breaks down to form the stable end-product TXB,. The role of metabolites of the lipoxygenase pathway is the focus of considerable current investigation. In previous studies, we have shown that lipid peroxidation can modulate AA turnover and PG synthesis in rabbit kidney medulla slices (2, 3) and that cyclooxygenase activity can be enhanced or inhibited by antioxidants depending on their type and concentration (4).

Date received 8 May I992 Date am-pled 10 July 1992

These observations have led us to speculate that lipid peroxidation may also affect the metabolism of AA via cyclooxygenase and lipoxygenase in platelets. In this paper. we describe our finding that tert-butyl hydroperoxide (t-BOOH) regulates platelet cyclooxygenase and lipoxygenase activities.

MATERIALS AND METHODS Materials TXB,, PGE,, PGD,. PGF,, AA. indomethacin. quercetin, t-BOOH and cumene hydroperoxide were obtained from Sigma ChemicaI Co, St. Louis, MO. USA and HHT, 12-HETE and 12-hydroperoxy5,8,10,16eicosatetraenoic acid (12-HPETE) were obtained from Cayman Chemical Co, Michigan, USA. l$Hydroxy-5,8.11.13-eicosatetraenoic acid (15-HETE) and 15hydroperoxy-5,8.11,13-eicosatetraenoic acid ( 15-HPETE) were obtained from Cascade Biochem Ltd, Berkshire, England and 9-antbryldiazomethane was obtained from Funakoshi Pharmaceutical Co, Tokyo, Japan. Desferrioxamine B mesylate was purchased from Ciba Geigy Japan. Hyogo. Japan. All other reagents were analytical grade.

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Preparation of platelets Blood was withdrawn into a 3.8% solution of trisodium citrate (9:1, v/v) from the abdominal aorta of male rabbits (2-2.5 kg) under sodium pentobarbital anaesthesia. Platelets were then collected by differential centrifugation. Whole blood was centrifuged for 10 min at 200 g at room temperature and the platelet-rich plasma was withdrawn from above the pelleted erythrocytes. After addition of EDTA (to a final concentration of 1 mM), the platelet-rich plasma was cooled to 0 “C and centrifuged at 2000 g for IO min. The platelet pellet was washed twice with 134 mM NaCl, 5 mM glucose, 15 mM Tris-HCl buffer, pH 7.4 (buffer A) containing 1 mM EDTA, and then resuspended in buffer A.

Incubation conditions and measurement of metabolites from AA The washed platelet suspension (3 x IO8 platelets) was preincubated for 5 min at 37 “C in 1 ml of buffer A with or without the indicated concentrations of indomethacin, quercetin, FeS04, t-BOOH, cumene hydroperoxide or 15-HPETE. Quercetin was firstly dissolved in ethanol and then diluted lOO-fold into the reaction mixture. Ethanol at 1% (v/v) had no effect on AA metabolism in platelets. AA (25 yM) was subsequently added to the platelet suspension, and the mixture was incubated at 37 “C for 5 min. The reaction was terminated by quickly adding an appropriate amount of 0.5 M-HCl to bring the pH of the reaction mixture to 3.0. The reaction mixture was then extracted with 3 ml of ethyl acetate. HHT and 12-HETE in the extracted lipid were simultaneously determined by a high-pressure liquid chromatographic (HPLC) method (5). HHT and 12-HETE were separated in normal-phase chromatography and simultaneously quantitated employing a Shimadzu model SPD-6A UV spectrophotometric detector, set at 234 nm, with an 16 ~1 flow cell. In the present study, HHT and 12-HETE were separated with a YMC packed column (A-003 type, 4.6 mm i.d. x 25 cm) eluted at 3 ml/min (Shimadzu model LC-6A pump) with n-hexane/isopropanol/acetic acid (98.4: I .6:00.04, v/v), A linear relationship between the height of peak and the amount of HHT or 12-HETE standard injected was observed over the range O500 pmoles. The detection limit was approximately 5 pmoles. The recovery of HHT and 12-HETE was 99.5 + 2.4% and 97.5 f 2.3%, respectively (means f SEM, n=5). The coefficient of variance for the assay of HHT and 12-HETE was 1.2% and 2.5%, respectively (n=5). TXB,, PGE,, PGD, and PGF?, in the extracted lipid were determined by a HPLC method using 9-anthryldiazomethane (ADAM) as described in our recent paper (6). By this method it was demonstrated that negligible amounts of PGE2, PGDz or PGF,, were found in the reaction mixture under all conditions. In some experiments, the amount of AA in the extracted lipid was measured by a HPLC method using ADAM (7). AA

esterified with ADAM (AA-ADAM) was separated in reverse-phase chromatography and simultaneously quantitated by employing a Shimadzu model RF-535 fluorescence spectrofluorometer (excitation 365 nm, emission 412 nm) with an 16 ~1 flow cell. The liquid chromatograph system used in this study was a Shimadzu model LC-6A system equipped with a YMC packed column (ODSA -303 type. 4.6 mm i.d. x 25 cm). The mobile phase consisted of acetonitrile/water (95:5, v/v). The flow rate was 1.2 ml/min. Peak heights were measured for the quantification of the AA-ADAM relative to the standard derivative prepared from authentic AA.

Statistics Results are means f SEM. Statistical determined by Student’s t-test.

significance

was

RESULTS AND DISCUSSION Washed rabbit platelets (3 x lO*/ml) metabolized exogenously added AA (25 PM) to I2-HETE, TXB, and HHT (Fig. 1). Under the experimental conditions described under Materials and Methods, in the absence of drugs. rabbit platelets produced 3-4 times more 12HETE than TXB? or HHT. Indomethacin is a selective cyclooxygenase inhibitor (8), while quercetin has been reported to inhibit lipoxygenase (9, IO). In order to confirm the validity of the present in vitro system, various concentrations of indomethacin and quercetin were added to the preincubation medium. When washed rabbit platelets were pretreated for 5 min with 1 FM-indomethacin followed by the addition of the AA substrate. the formation of products of cyclooxygenase (TXB, and HHT) was completely inhibited, but the production of lipoxygenase metabolite (12-HETE) was not inhibited (Fig. 1A). In

Fig. 1 Effects of indomethacin (A) and quercetin (B) on the formation of cyclooxygenase and lipoxygenase products from AA in washed platelets. Platelets were preincubated with various concentrations of indomethacin or quercetin for 5 min at 37 “C prior to the incubation with AA (25 PM) for 5 min. Other details were as described in Materials and Methods. Results are mean values I SEM (n=6). *p < 0.01 compared with the corresponding value in the absence of indomethacin or quercetin.

tert-Butyl Hydroperoxide and AA Metabolism in Platelets

contrast, preincubation of the platelets with quercetin (l-100 PM) caused a concentration-dependent inhibition of 12-HETE production (Fig. 1B). The formation of TXB, and HHT was not inhibited by preincubation with quercetin at concentrations of up to 10 ,uM, but was inhibited by 100 FM-quercetin. A similar tendency was observed after pretreatment of epidermai lipoxygenase and cyclooxygenase with quercetin (IO). Thus, these data demonstrate the capacity of the present in vitro system to simultaneously detect changes in cyclooxygenase and lipoxygenase enzyme metabolism by platelets. We have previously reported that Fe’+ has a powerful stimulatory effect on the lipid peroxidation of rat kidney cortex slices (11) and rabbit kidney medulla slices (2). We have also shown that lipid peroxidation induced by Fe:+ inhibits PCE, generation in rabbit kidney medulla slices (2, 3).Therefore, we first examined the influence of Fe’+ on the production of 12-HETE, TXB? and HHT in washed rabbit platelets (Fig. 2). The formation of 12HETE was inhibited by preincubation of the platelets with Fe?+ at concentrations ranging from lo-500 PM. The effect of Fe)+ was concentration-dependent. On the other hand. Fe?+ at 10 or 30 FM tended to stimulate the formation of TXB, and HHT, but the stimulation was not statistically significant. Fe’+ did not affect the formation of TXBl and HHT at concentrations between 40 and 100 PM, whereas it significantly inhibited the formation at 500 PM. Undoubtedly, in the presence of Fe?+, AA is susceptible to peroxidation. Depletion of the AA substrate results in inhibition of AA metabolism by platelets. However. this simple explanation does not seem valuable in the case of lower concentrations of Fe?+ since Fe’+ reduces the formation of 12-HETE without inhibiting the production of TXB, and HHT at concentrations of 100 PM and below. These observations suggest that lipid peroxidation induced by Fe” is connected closely with the lipoxygenase pathway in platelets. It has been reported that hydroperoxides can affect the synthesis of PGs by cyclooxygenase from sheep ve-

sicular glands or calf aortae at several levels. Very low concentrations of hydroperoxides are required to activate cyclooxygenase. and in their absence, there is very little enzyme activity ( 12). On the other hand, higher concentrations of these substances can inhibit cyclooxygenase ( 13. 14). From this viewpoint. we speculated that the intracellular concentration of hydroperoxides might be an important factor in regulating AA metabolism via cyclooxygenase and lipoxygenase in platelets. In order to test this hypothesis, we examined the effect of tBOOH on the formation of 12-HETE, TXB, and HHT in washed rabbit platelets. As shown in Figure 3, preincubation of the platelets with t-BOOH (2-50 FM) showed a dose-dependent inhibition of 12-HETE production. At a concentration of 10 PM, t-BOOH suppressed the production of 12-HETE to 10%) of control. On the other hand, the formation of TXB, and HHT was increased significantly by t-BOOH at concentrations of 8 FM and below. with a maximum increase of 142% compared with control. At concentrations of t-BOOH higher than those causing maximal stimulation a reversal of effect was seen. resulting in inhibition of TXB, and HHT formation at SO FM. Thus, t-BOOH inhibited both the cyclooxygenase and lipoxygenase pathways at a concentration of SO AM. In addition, the amount of AA remaining after incubation was quantified by HPLC. In the control experiment, when the platelets were preincubated in the absence oftBOOH followed by the addition of AA (25 nanomoles), 4.3 f 0.2 nmole AA was detected (mean & SEM, n=5). Raising the t-BOOH concentration from 0 to 8 FM increased the amount of AA from 4.3 + 0.2 to 10.8 + 0.5 nmole. and even at a concentration of t-BOOH of SO FM 10.7 + 0.4 nmole AA remained (mean + SEM, n=.5). This result lessens the possibility that t-BOOH (50 PM)induced reduction of 12-HETE. TXB, and HHT formation can be ascribed to decreased availability of AA by

Effects of t-BOOH on the formation of I?-HETE, TXB, and platelets. Platelets were preincubated with various concentrations of t-BOOH for 5 min at 37 “C prior to the incubation with A (25 PM) for 5 min. Other details were as described in Materials and Methods. Results are mean values k SEM (n=6). *p < 0.02 compared with the corresponding value in the absence oftBOOH. **p < 0.01 compared with the corresponding value in the absence of Fig. 3

HHT in washed

Fig. 2 Effects of Fe” on the formation of I2-HETE, TXBzand HHT in washed platelets. Platelets were preincubated with various concentrations of Fe?+ for 5 min at 37°C prior to the incubation with AA (25 PM) for 5 min. Other details were as described in Materials and Methods. Results are mean values + SEM (n=6). *p < 0.01 compared with the corresponding value in the absence of Fe”.

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t-BOOH.

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its peroxidative action. Additionally, because we found that the cyclooxygenase was saturated with 5 PM AA in preliminary experiments, the enhancement of TXB, and HHT formation elicited by t-BOOH (2-8 PM) was probably not the result of increased availability of AA by inhibition of the lipoxygenase pathway. So, it seems likely that cyclooxygenase activity in platelets can be enhanced or inhibited by t-BOOH depending on the concentration. Platelet lipoxygenase transforms AA to the labile precursor of IZHETE, 12-HPETE. It is rapidly rapidly converted to 12-HETE by HPETE peroxidase activity in intact platelets and platelet homogenates (15, 16). To investigate whether t-BOOH inhibits the conversion of 12-HPETE to 12-HETE, washed rabbit platelets were incubated with 12-HPETE instead of AA (Table 1). When 12-HPETE (8 nmole) was incubated for 5 min at 37 “C in the absence of the platelets, only a small amount of 12-HETE (< 0.1 nmole) was recovered. Control platelets converted 5 1% of added 12-HPETE to 12-HETE after a 5-min incubation at 37 “C. Preincubation of the platelets with t-BOOH (6 FM) for 5 min at 37% did not show any inhibition of the conversion of 12-HPETE to 12-HETE. This result suggests that t-BOOH inhibits 12-HETE formation by inhibiting the lipoxygenase step rather than by affecting HPETE peroxidase activity. To further compare the effect of t-BOOH on the formation of 12-HETE, TXB2 and HHT with other hydroperoxides, cumene hydroperoxides and 15-HPETE were subjected to the same experimental protocols as applied to t-BOOH. When cumene hydroperoxide was added to the preincubation mixture of platelets, results identical to those with t-BOOH were obtained (results not shown). As shown in Figure 4, 15-HPETE showed a dose-dependent inhibition of 12-HETE formation, but the inhibitory effect was kept low compared with t-BOOH at concentrations of 8 PM and below (15HPETE, 19-75% inhibition; t-BOOH, 43-88% inhibition). In contrast to the stimulatory effect of t-BOOH (2-8 PM) on the formation of TXB, and HHT, 15HPETE induced no change in the formation at concentrations of up to 6 ,uM. At concentrations of 8 PM or more, the formation of TXB, and HHT was inhibited significantly by 15-HPETE. It is possible that the differences between the effects on 12-HETE, TXB, and Table 1 I2-HETE

Effects of t-BOOH on the conversion

Preincubation

12.HETE formed (nanomoles)

of 12-HPETE to

% Conversion of 12-HPETE to I 2.HETE

Buffer alone Platelets Platelets + t-BOOH (5 ,uM)

0.095 i 0.003 4.106 + 0.141 4.353 * 0.151

1.2 51.3 54.4

Washed platelet suspensions (3 x 10R/ml) were preincubated for 5 min at 37 “C in 1.O ml of 134 mM NaCI, 5 mM glucose. 15 mM Tris-HCI buffer (ph 7.4) in the absence and the presence of t-BOOH (6 PM), and then 12-HPETE (8 nanomoles) was added for 5 min. Results are mean values + SEM (n=3)

Fig. 4 Effect of 15-HPETE on the formation of 12-HETE. TXBz and HHT in washed platelets. Platelets were preincubated with various concentrations of 15-HPETE for 5 min at 37 “C prior to the incubation with AA (25 ,uM) for 5 min. Other details were as described in Materials and Methods. Results are mean values f SEM (n=3) *p < 0.05 compared with the corresponding value in the absence of 15-HPETE. **p < 0.01 compared with the corresponding value in the absence of 15-HPETE.

HHT formation of t-BOOH and 15-HPETE may be related to differing rates of metabolism in platelets, because we observed the rapid conversion of 15-HPETE to 15-HETE in this experiment. (This product was identified by comparison of the retention time with that of 15-HTE standard). Vanderhoek et al (17) reported that 15-HETE is a platelet lipoxygenase inhibitor but is less potent than 15-HPETE. In addition, during its conversion to 15-HETE, 15-HPETE produces hydroxy radicals (18) that may inactivate cyclooxygenase (19). It is not certain to what extent the parent compound or metabolites, or both, of these relatively rapidly metabolized components may have contributed to the effects seen. However, the data showing the higher sensitivity of platelet cyclooxygenase and lipoxygenase to t-BOOH compared to 15-HPETE at low concentrations may have important physiological implications. Next, to investigate the mechanism by which t-BOOH inhibits platelet lipoxygenase, we examined the effect of the addition of 1 ,uM-indomethacin during the preincubation of washed rabbit platelets with t-BOOH (Table 2). The inhibitory effect of t-BOOH (2 /.fM) on 12-HETE formation could not be blocked by the addition of indomethacin at a concentration where the cyclooxygenase pathway was inhibited, indicating that t-BOOH inhibits the lipoxygenase independently of its Table 2 formation

Effects of t-BOOH in the presence of indomethacin of i2-HETE, TXB, and HHT in washed platelets

Pretreatment Control lndomethacin (1 PM) t-BOOH (2 /iM) Indomethacin + t-BOOH

on the

12.HETE TXB? HHT (nanomoles/3 X 10s platelets) 7.691 + 0.384 7.948 f 0.396 4.383F0.219 2.105 k 0.105

2.660 + 0.129 n.d. 3.695iO.181 0.246 f 0.013

2.052 f 0.100 n.d. 2.865kO.142 0.084 + 0.03

Platelets were preincubated with and without indomethacin (I PM) and t-BOOH (2 ,uM) for 5 min at 37’C prior to the incubation with AA (25 PM) for 5 min. Other details were as described in Materials and Methods. Results are mean values + SEM (n=5). n.d.. not determined.

tert-Butyl Hydroperoxide Table 3 Effects of t-BOOH in the presence of desferrioxamine formation of 12-HETE. TXB2 and HHT in washed platelets 13.HETE (nanomoles/.i

Pretreatment Control Desferrioxamine (2 mM) t-BOOH (3 PM) Desferrioxamine + t-BOOH

7.910 7.333 4.534 2.611

+ 0.394 ? 0.359 * 0.226 +0.130

and AA Metabolism

in Platelets

263

on the

HHT TXB: x 10” platelets)

2.727 2.528 3.817 3.770

f 0.129 + 0.130 +0.190 rf-0.190

2.109 1.945 2.939 2.799

* * * *

0.103 0.094 0.145 0.138

Platelets were preincubated with and without desferrioxamine (2 PM) and tBOOH (2 PM) for 5 min at 37 “C prior to the incubation with AA (25 FM) for S min. Other details were as described in Materials and Methods. Results are mean values + SEM (n=7).

ability to stimulate the cyclooxygenase activity. It can be conceived that the inhibition by t-BOOH of platelet lipoxygenase is due to a direct effect of t-BOOH on the enzyme. When t-BOOH and indomethacin were added together, 12-HETE formation was inhibited further as compared with t-BOOH alone. We did not analyse this finding in detail, but the most feasible explanations are that in the presence of indomethacin t-BOOH is not used for activation of the cyclooxygenase, and that larger amounts of t-BOOH act on the lipoxygenase. Desferrioxamine is an effective iron-chelating agent. Intracellular ferric iron derived from either ferritin or transferrin is reduced to ferrous iron by superoxide anions (20). The ferrous iron then reacts with hydrogen peroxide to form hydroxy radicals. Such a sequence has been referred to as an iron-catalysed Haber-Weiss reaction (21). Previously it was shown that desferrioxamine effectively removes iron from ferritin (22. 23). Gutteridge et al (24) reported that desferrioxamine binds Fe (III) tightly so that it cannot be reduced by superoxide anions, and hence it inhibits the formation of hydroxy or alkoxy radicals and enhances the concentration of hydrogen peroxide or hydroperoxides. Therefore. we determined the effect of t-BOOH in the presence of desferrioxamine on the production of 12-HETE. TXB? and HHT in washed platelets (Table 3). Preincubation of the platelets with desferrioxamine (2 mM) showed no significant effect on the formation of 12-HETE. TXB, and HHT. The addition of desferrioxamine made the inhibitory effect of t-BOOH (2 ,uM) on I2-HETE formation more pronounced. The data were interpreted as indicating that the inhibitory effect of t-BOOH on platelet lipoxygenase was due to the hydroperoxy moiety. In contrast to the above observations, Siegel et al (25) have reported that 12-HPETE (a species of hydroperoxide) stimulates platelet lipoxygenase activity. One explanation for this discrepancy could be that there is a unique interrelationship between 12-HPETE and platelet lipoxygenase that is not common to either t-BOOH or the other hydroperoxides studied. Such an interrelationship is consistent with the fact that 12-HPETE alone of these hydroperoxides is a potential intermediate in the platelet lipoxygenase pathway. Our present results suggest that hydroperoxides produced during lipid peroxidation strongly inhibit pla-

telet lipoxygenase activity (Figs 2 & 3) and that the hydroperoxy functional group is a prerequisite. The mechanism by which the hydroperoxides inhibit the lipoxygenase of platelets is presently unknown. However, sine it has been established that platelet lipoxygenase contains non-heme iron (26), the mechanism for this inhibition appears to be different than the activation of cyclooxygenase (which contains heme iron) (12, 27) and may involve the altered redox-balance of the nonheme iron atom of the lipoxygenase. The polyunsaturated fatty acids in phospholipids of platelets form hydroperoxide intermediates during lipid peroxidation. The results of this work suggest that these hydroperoxides play an important role in the control of both platelet cyclooxygenase and lipoxygenase activities. These observations provide new insight into factors controlling the production of the metabolites derived from AA in platelets.

References 1. Hamberg M, Samuelsson B. Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Nat1 Acad Sci USA 1974: 71: 3400-3404 2. Fujimoto Y. Fujita T. Effects of lipid peroxidation on prostaglandin synthesis in rabbit kidney medulla slices. Biochim Biophys Acta 1982; 710: 82-86 3. Fujimoto Y. Tanioka H, Keshi I, Fujita T. The interaction between lipid peroxidation and prostaglandin synthesis in rabbit kidney-medulla slices. Biochem J 1983: 212: 167-171 4. Fujita T. Fujimoto Y. Tanioka H. Antioxidant effects on prostaglandin synthesis in rabbit kidney medulla slices. Experientia 1982; 38: 1472 5. Beving H. Determination of platelet-megakaryocyte regeneration time in painters occupationally exposed to organic solvents. J Chromatogr 1986; 382: 67-77 6. Fujimoto Y. Nakatani E. Minamino H, Takahashi M. Sakuma S, Fujita T. Effective use of rabbit gastric antral mucosal slices in prostaglandin synthesis and metabolism studies. Biochim Biophys Acta 1990: 1044: 65-69 1. Nimura N. Kinoshita T. Fluorescent labeling of fatty acids with 9-anthryldiazomethane (ADAM) for high performance liquid chromatography. Anal Lett 1980: 13: 191-202 8. Flower R J. Drugs which inhibit prostaglandin biosynthesis. Pharmacol Rev 1974: 26: 33-67 9. Hope W C. Welton A F. Fiedler-Nagy C. BatulaBernard0 C. Coffey J W. In vitro inhibition of the biosynthesis of slow reacting substances of anaphylaxis (SRS-A) and lipoxygenase activity by quercetin. Biochem Pharmacol 1983: 32: 367-37 I 10. Nakadate T. Yamamoto S, Aizu E, Kato R. Inhibition of

264

Prostaglandins

Leukotrienes

and Essential Fatty Acids

12-O-tetradecanoylphorbol-13increase in vascular permeability in mouse skin by lipoxygenase inhibitor. Jpn J Pharmacol 1985: 38: 161-168 11. Fujimoto Y, Fujita T. Effect of lipid peroxidation on paminohippurate transport by rat kidney cortical slices. Br J Pharmacol 1982; 76: 373-379 12. Hemler M E, Lands W E M. Evidence for a peroxideinitiated free radical mechanism of prostaglandin biosynthesis. J Biol Chem 1980; 255: 62536261 13. Nugteren D H, Hazelhof E. Isolation and properties of intermediates in prostaglandin biosynthesis. Biochim Biophys Acta 1973; 326: 448461 14. Powell W S, Granvelle F. Metabolism of eicosapentaenoic acid by aorta: formation of a novel 13hydroxylated prostaglandin. Biochim Biophys Acta 1985; 835: 202-211 15. Siegel M 1. McConnell R T, Cuatrecasas P. Aspirin-line drugs interfere with arachidonate metabolism by inhibition of the 12-hydroperoxy-5.8,10.14eicosatetraenoic acid peroxidase activity of the lipoxygenase pathway. Proc Nat1 Acad Sci USA 1979; 76: 3774-3778 16. Siegel M 1, McConnell R T. Porter N A, Cuatrecasas P. Arachidonate metabolism via lipoxygenase and 12Lhydroperoxy-5&10,14_icossatetroenoic acid peroxidase sensitive to anti-inflammatory drugs. Proc Nat1 Acad Sci USA 1980: 77: 308-312 17. Vanderhoek J Y. Bryant R W. Bailey J M. 15.Hydroxy5.8.1 I. 13-eicosatetraenoic acid. A potent and selective inhibitor of platelet lipoxygenase. J Biol Chem 1980: 255: 5996-5998 18. Ham E A, Egan R W, Soderman D D, Gale P H. Kuehl F A Jr. Peroxidase-dependent deactivation of prostacyclin

synthetase. J Biol Chem 1979: 2191 19. Egan R W, Paxton J, Kuehl F A Jr. Mechanism for irreversible self-deactivation of prostaglandin synthetase. J Biol Chem 1976: 25 1: 7329-7335 20. Starke P E. Farber J L. Ferric iron and superoxide ions are reguired for the killing of culture hepatocytes by hydrogen peroxide. Evidence for the participation of hydroxyl radicals formed by an iron-catalyzed HaberWeiss reaction. J Biol Chem 1985: 260: 10099-10104 21. McCord J M. Day E D Jr. Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett 1978; 86: 139-142 22. Octave J N, Schneider Y-J. Crichton R R. Trouet A. Iron mobilization from cultured hepatocytes: effect of desferrioxamine B. Biochem Pharmacol 1983; 32: 3413-3418 23. Bridges K R. Hoffman K E. The effects of ascorbic acid on the intracellular metabolism of iron and ferritin. J Biol Chem 1986: 14273-14277 24. Gutteridge J M C. Richmond R. Halliwell B. Inhibition of the iron-catalysed formation of hydroxyl radicals from superoxide and of lipid peroxidation by desferrioxamine. Biochem J 1979; 184: 469-472 25. Siegel M I. McConnell R T, Abrahams S L, Porter N A, Cuatrecasas P. Regulations of arachidonate metabolism lipoxygenase and cyclooxygenase by 12-HPETE, the product of human platelet lipoxygenase. Biochem Biophys Res Commun 1979; 89: 1273-1280 26. Aharony D, Smith J B. Silver M J. Human platelet lipoxygenase reguires ferric iron. Fed Proc 1980: 39: 4243 37. Kulmacz R J. Lands W E M. Prostaglandin H synthase. Stoichiometry of heme cofactor. J Biol Chem 1984: 259: 6358-6363