PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS F’rostaglandins Leukotrienes and Essential 0 Longman Group UK Ltd 1992

Fatty Acids

t1992)

47. 187-191

Influence of N-phenyllinoleamkde from Toxic Oil Samples on the Lipoxygenase Metabolism of Exogenous Arachidonic Acid in Mouse Peritoneal Macrophages G. Bioque, 0. Butbena, G. Gbmez, J. RoseWCatafau

and E. Gelpi

Molecular Pathology Unit, CID-CSIC, Jordi Girona 18-26. 08034 Barcelona, Spain (Reprint requests to JR-C) ABSTRACT. N-phenyllinoleamide (NPLA) is a useful marker for adulterated oil samples associated with cases of toxic oil syndrome (TOS). To date, NPLA has not reproduced the human poisoning episode in experimental animal models and, thus, its pathological role in the syndrome remains controversial. The present report describes the effect of NPLA on the lipoxygenase metabolism of exogenous arachidonic acid (AA) in mouse peritoneal macrophages (MPM). Results show that MPM cells exposed to 1mM NPLA for 2 h, when subsequently incubated with exogenous 3H-AA, undergo a significant increase in the biosynthesis of 3H-12hydroxyeicosatetraenoic acid (3H-!2-HETE) whereas levels of 3H-15-HETE are relatively stable. These data indicate that NPLA selectively potentiates the lipoxygenase metabolism of exogenous AA, supporting the possible implication of lipid peroxidative processes in the ethiopathology of TOS, although the relatively high NPLA concentration required ‘in vitro’ makes it unlikely that this xenobiotic could be directly related to human toxicity. -

INTRODUCTION

oxidative AA metabolism increasing the biosynthesis of LTs (7), prostaglandins (PGs) and thromboxane (TX)

The fatty acid anilides (FAA) are to date the only extraneous compounds detected in relatively high amounts (10-1000 ppm) in adulterated cooking oils which intoxicated more than 20 000 people, and caused over 600 deaths in a unique epidemic disease known as the toxic oil syndrome (TOS) (1). FAA are considered to be helpful analytical markers of toxic case related oils (2, 3), but to date they have not satisfactorily reproduced the syndrome in laboratory animals. Furthermore, in the early days of research on the causes of TOS, synthethic anilides were found to be almost devoid of ‘in vitro’ toxicity (4) and no correlation has been established between claims of ‘in vitro’ cell toxicity and anilide content in the oils. Nevertheless, it has been argued that some of the clinical signs of TOS could be associated to lipid peroxidative processes, mediated by FAA (5, 6). In this sense, bioactive metabolites resulting from the biological oxidation of arachidonic acid (AA) such as the leukotrienes (LTs) (7,8) have been implicated in the etiopathogenesis of TOS, and it has also been described that the anilide of linoleic acid or N-phenyllinoleamide (NPLA) enhances

B, (9). On account of these results, we therefore investigated the effects of NPLA on the exogenous lipoxygenase metabolism of AA in mouse peritoneal macrophages (MPM). The aim of this work was thus to ascertain whether FAA, as markers of epidemiologically validated toxic oils, could have a direct influence on lipid peroxidative processes in an ‘in vitro’ model of study in a similar way as we had done before with the cyclooxygenase pathway (9). For this reason, we have now measured the production of (3H-12-hydroxyeicosatetraenoic acid (3H-12-HETE) and 3H-15-HETE from exogenously provided ‘H-AA in MPM preexposed to NPLA.

MATERIAL

AND METHODS

Cell isolation Mouse macrophages were collected by peritoneal lavage with Dubelco’s phosphate buffer saline (PBS). The cells were washed by sedimentation (20 min. 400 xg, 16°C) and resuspended in minimum essential medium Eagle (modified) with Earle’s salts (EMEM), supplemented with L-glutamine (2mM), sodium pyruvate (ImM), non essential aminoacids mixture (l%), penicillin and streptomycin (100 Ill/ml) and fetal bovine serum (10%)

Date received 1 April 1992 Date accepted 14 May 1992 187

188

Prostaglandins Leukotrienes and Essential Fatty Acids

(Flow Laboratories. McLean, VA, USA). Cellular density was adjusted at 5 x I06/m! and 2m! a!iquots were distributed into 35 mm plastic culture dishes (Costar, Cambridge, MA, USA), where they were kept for 2 h at 37°C. Finally, nonadherent cells were removed by four washes with PBS. This method, described by Dimitriu (lo), enables the collection of viable macrophages at 99% purity.

Cell incubations Preincuhation with NPLA NPLA, synthesized as previously described by us (1 I). was prepared at different concentrations ( I. 0.1,O.O 1 mM) in EMEM by repeated sonication in an ice bath (12) for inmediate incubations at different times (from 5 min to 2 h at 37°C). Finally, NPLA exposed macrophages were washed four times with 2 ml of PBS for NPLA removal and subsequent reincubation with ‘H-AA. 3H-AA incubations Unexposed and NPLA exposed macrophages cubated at different times (from 2.5-60 min with 2 ml of PBS containing 0.5 pCi ?H-AA activity 60 Ci/mmol, New England Nuclear, MA, USA). After the incubation period, the cubate supernatants in each we!! were collected sequent extraction and HPLC analyses.

were inat 37°C) (specific Boston, 2 ml infor sub-

Lipoxygenase inhihition Cells unexposed and NPLA exposed were incubated for 2 h at 37°C. After washing out the NPLA, they were further incubated as above with ‘H-AA (0.5 pCi) in PBS for 8 min at 37°C. In both phases of incubation esculetin (1 mM) was added to the medium. The resulting supernatant was collected for extraction and HPLC assay.

Extraction

and HPLC procedures

The incubates were acidified at pH 3.15 and extracted four times by addition of ethyl acetate (1 ml). After evaporation of the organic extracts to dryness, the dry residues were resuspended in chromatographic eluent and analyzed by reversed phase HPLC using ‘on line’ radioactivity monitoring techniques (13). The HPLC separations were carried out by using a 5 pm(25 x 0.46 cm) Ultrasphere C 18 column (Beckman Instrument, San Ramon, CA, USA). The mobile phase was a mixture of methano!:water:trifluoroacetic acid:triethy!amine (80:20:0.1:0.05) run isocratically for 14 min at a flow rate of 1.5 ml/min and under a linear gradient to 100% methanol from I4 min to 17 min ( 14).

MPM with NPLA, prior to the addition of AA. For this purpose, the cells were exposed to 0.1 mM NPLA for different periods of time ranging from 5 min to 2 h. In a!! cases, after washing out the NPLA from the MPM monolayer with PBS, these NPLA pretreated cells were further exposed to labelled AA (3H-AA, 05 uCi/we!!) for I h at 37°C. From the radioactivity profile of the incubation medium, the production of 15- and I?-HETE as we!! as residual unmetabolized AA could be determined. Positive HETE identification was established with authentic standards and from the disappearance of these peaks when a iipoxygenase inhibitor, such as esculetin. was added to the incubation medium (see below). Figure 1 shows the variation of these compounds with NPLA incubation time. According to these data, maximum and stable levels of HETEs are achieved between l-2 h of preincubation with NPLA. Thus, optimum pretreatment time with NPLA was set at 2 h for all further experiments (7). At this time, a study by transmission electron microscopy proved that there were no morphological differences between MPM exposed to NPLA and the corresponding unexposed controls (9). Cell viability was also assayed by the trypan blue exclusion method and was found to be higher than 95%. There was no evidence of ‘H-l 5- or jH-IZ-HETE production in cell free incubations. As regards to the optima! time of MPM incubation with exogenous AA, the time course of the production of these lipoxygenase metabolites was also studied. For this

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AND DISCUSSION

The time course effect of NPLA on the metabolism of exogenous AA in MPM was studied by preincubating

NPLA

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2h time

Fig. I Time course effect of NPLA preexposure (0. I mM) on the biosynthesis of Ill-(open circles) and I5-HETE from exogenous ‘HAA (incubation time with 3H-AA, I h) in mouse peritoneal macrophages. Results are the mean of two independent experiments.

Influence of N-phenyliinoleamide

purpose, cells preexposed to 0.1 mM NPLA (2 h at 37°C) as well as control cells were incubated with “HAA (0.5 pCi/well) for periods of time increasing from 2.5 to 60 min. The kinetics of 3H- 15 and 3H-12-HETE generation under these conditions are shown in Figure 2. As illustrated, levels of 12-HETE reach a peak at 510 min (Fig. 2A) whereas peak levels corresponding to 15-HETE were reached at 5-15 min (Fig. 2B). In 1 h, 12-HETE practically disappeared while 15-HETE levels appeared to be more stable. Thus, optimum incubation time with 3H-AA was set at 8 min which would be in line with data provided in the literature by other authors (15). With NPLA exposure and AA incubation times optimized, as described above, a study of the lipoxygenase biosynthetic activity of MPM exposed to NPLA vs nonexposed controls was undertaken, as follows. Figure 3 shows the radioactivity profiles from MPM preexposed to NPLA (0.1 mM, 2 h at 37°C) and then incubated with H-AA for 8 min (A) and I h (B). In the latter case, a highly significant reduction in 12-HETE (peak 4) after 1 h incubation is concomitant to an increase in the first eluting band (peak 1). This broad peak is due to the coelution of higher polarity components. The GC/MS analysis shows the presence in peak 1 of

on Lipoxygenase Metabolism of Exogenous AA

a mixture of trihydroxyeicosatrienoic acids, possibly arising from oxidation of 12-HETE. Peak 2 was also identified as a family of vicinal epoxyhydroxyacids derived from AA through the corresponding hydroperoxy eicosatetraenoic intermediates (16). The dose-response effect of NPLA on lipoxygenasic exogenous 3H-AA metabolism was also studied. For this purpose, MPM were exposed to varying NPLA concentrations for 2 h at 37°C and then the cells were incubated with 3H-AA for 8 min at 37’C. Results showed that whereas ‘H-15-HETE was unchanged, there was a moderate but significant increase in )H-1ZHETE at the 1 mM NPLA dose (Fig. 4). As also illustrated in this Figure, a specific lipoxygenase inhibitor, such as esculetin, resulted in a highly significant reduction in both 15- and 12-HETE levels, further validating this assay model. The effect of NPLA on 12-HETE biosynthesis (Fig. 4) is in line with previously reported data showing an increased biosynthesis of 6-keto-PGF,, and TXB, from exogenous AA, in MPM also preexposed to NPLA for 2 h (9). Results along this line have been reported previously by Gil and coworkers (7), who studied the mechanism whereby NPLA generates AA in polymorphonuclear leukocytes (PMNs). Their results indicate that NPLA

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Kinetics of ‘H- 12-HETE (A) and JH- 15HETE (8) production from exogenous ‘H-AA by MPM previously unexposed (closed circle) (n=6) and exposed for 2 h to a 0.1 mM NPLA dose (stars) (n=2). Results are expressed as mean f SD.

189

Time

Fig. 3

(mid

HPLC radioactivity profile of the products generated by MPM from exogenous ‘H-AA in 8 min (A) and 1 h (B) incubation time, after previous pretreatment with NPLA (0.1 mM) for 2 h. 1: unresolved trihydroxyeicosatrienoic acids; 2: unresolved epoxy eicosatrienoic acids; 3: jH- 15HETE; 4: jH- 12-HETE.

190

Prostaglandins

Leukotrienes

and Essential Fatty Acids

could favor the activation of phospholipase A2 with the subsequent release of free AA which would then be metabolized to HETEs. These compounds, together with their fatty acid hydroperoxide precursors have been implicated in the mechanism of action of the etiopathogenic agent in TOS (8). However, on account of the results of this work, and considering also the negative results to date on animal models, it would seem that the direct toxicity of NPLA is low (as indicated above, cell viability is higher than 95% even at the 1 mM dose), although it would still be tempting to speculate that NPLA could engage or potentiate mechanisms of immune and inflammatory responses, mediated by AA metabolites. It is also noteworthy that the effects of NPLA become significant at the 1 mM dose, so that biological significance might be far from being of relevance to human studies. This consideration would apply to most of the experimental studies on biochemical effects of fatty acid anilides (7, 9, 12). In conclusion, NPLA potentiates the biosynthesis of the major lipoxygenase metabolite in MPM. Although the resultant increase of 12-HETE could be associated to some of the clinical manifestations of TOS, the concentration of NPLA required ‘in vitro’ (1 mM) indicates that its effect on arachidonic acid metabolism is unlikely related to a still not proven ‘in vivo’ toxicity of NPLA in toxic oils. Acknowledgements Financial support from the Fondo de Investigaciones through the projects 88/271 and YO/1261 is gratefully

Sanitarias (FIS) acknowledged.

References

Fig. 4 Bar plot depicting the percentage of radioactivity (mean f SEM) as ‘H-12-HETE (A) and ‘H-15-HETE (B) recovered in the supematant of MPM cells incubated with ‘H-AA for 8 min: 1) NPLA unexposed cells; 2 h incubation (control) (n=6). II) 0.01 mM NPLA preexposed cells; 2 h incubation (n=5). III) 0. I mM NPLA preexposed cells; 2 h incubation (n=5). IV) I mM NPLA preexposed cells; 2 h incubation (n=5). V) Control with 1 mM esculetin; 2 h incubation (n=5). VI) 1 mM NPLA and esculetin preexposed cells; 2 h incubation (n=5). *p