The effect of individual unsaturated fatty acids on the release of tumour necrosis factor (TNF) and interleukin 6 (IL6) was investigated in thioglycollate-induced rat peritoneal macrophages. The intracellular mechanisms associated with the changes of cytokine production in response to fatty acids were also studied. Incubation of macrophages with 100 microM docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) increased TNF (21% and 15% respectively) and IL6 (69% and 40% respectively) production. Linoleic acid (LA) diminished TNF production by 16%. At 100 microM oleic acid (OA), LA and EPA concentration an increase in macrophage adenylate cyclase activity (110%, 72% and 39% respectively) and a decrease (14%) in the presence of DHA was observed. PGE2 production in the presence of 100 microM DHA was reduced by 36%, whereas in the presence of 100 microM LA an increase (75%) was observed. Phospholipase A2 (PLA2) activity was also found to be modified in the presence of EPA and DHA at 50 microM (20% and 60% respectively) and 100 microM (34% and 62% respectively) concentrations. The activities of both protein kinase A (PKA) and protein kinase C (PKC) were effected by the different fatty acids. At 50 microM all fatty acids suppressed PKA activity except OA which enhanced PKA activity by 14%. At 100 microM fatty acid concentration, EPA suppressed PKA activity by 40%. PKC activity was enhanced by LA and OA, by 18% and 21% respectively. However, at 100 microM EPA and DHA, PKC activity was suppressed by 37% and 17% respectively, whereas PKC activity was enhanced by 146% in the presence of 100 microM LA. These results show for the first time that unsaturated fatty acids have an effect on macrophage PLA2 activity and that PGE2 may be a potent modulator of IL6 production. From these studies it is tempting to speculate that macrophage TNF and IL6 release may, in part, occur via a PKC and PKA independent pathway and that PLA2 activity and PGE2 concentration are inversely related to production of TNF and IL6.

This study has investigated the protein changes in rat liver elicited by a group of model hepatotoxicants, methapyrilene, cyproterone acetate and dexamethasone and offers a compelling argument in support of the use of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry for the identification of compound specific biomarkers. The different treatments caused distinct changes to the rat liver proteome. Many of the protein changes could be associated with the known pharmacological and toxicological mechanisms of action of these drugs, whereas for other proteins, the rationale behind the alterations was less obvious. Furthermore, these changes can be used to classify the treatments with a view to utilising them as 'molecular signatures' to further our understanding of less well studied drugs such as SKF-106686 (an adrenoreceptor agonist). This approach has the potential for opening up new avenues for the exploration of molecular mechanisms of toxicity. This paper has explored the feasibility of proteomics to provide valuable information on the biochemical consequences elicited by hepatototoxic drugs.

Peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor superfamily and are intimately involved in lipid metabolism and energy homeostasis. Activation of these receptors in rodents can lead to hepatomegaly and ultimately hepatic carcinogenesis although the mechanisms by which these processes occur are poorly understood. To further our understanding of these processes and to discriminate between different PPAR mediated signalling pathways, a proteomic approach has been undertaken to identify changes in protein expression patterns in Sprague Dawley rat liver following dosing with a PPARalpha agonist (Wyeth 14643), a PPARgamma agonist (Troglitazone) and a compound with mixed PPARalpha/gamma agonist activity (SB-219994). Using one-and-two-dimensional electrophoresis of tissue lysates a diverse range of protein abundance changes was observed in these tissues. Whilst a number of these proteins have PPAR response elements (PPREs) in their respective promoters, another group was detected whose expression has been documented to be sensitive to peroxisome proliferator administration. Most notably within these groups, proteins involved in lipid catabolism displayed increased expression following drug administration. A further subset of proteins, with less obvious biological implications, also showed altered expression patterns. Where available, sequences upstream of the coding regions of genes not previously known to have PPREs were searched with positional consensus matrices for the presence of PPREs in an attempt to validate these changes. Using such an approach putative PPARgamma and PPARdelta response elements were discovered upstream of the tubulin beta coding region. There was limited overlap in observed protein abundance changes between the three groups, and where this was the case (cytosolic epoxide hydrolase, peroxisomal bifunctional enzyme, hydroxymethyl glutaryl CoA, synthase, long chain acyl-CoA thioesterase), expression of these proteins had previously been shown to be under the control of PPAR activity.

Principal component analysis (PCA) has been used to analyse mass spectral peptide profiles obtained from the enzymatic digestion of standard protein mixtures. Scores and loadings plots clearly revealed peptide fragments that differentiated one protein mixture from another. Peptide map search results identified with a high degree of certainty any additional proteins in these mixtures. As a proof-of-concept this methodology was applied to hepatic protein mixtures obtained from rats treated with two hepatotoxic compounds: methapyriline and SB-219994. Liver proteins were extracted, pre-separated by one-dimensional polyacrylamide gel electrophoresis, subjected to tryptic digestion and analysed by mass spectrometry. Two up-regulated proteins, glutathione S-transferase with methapyrilene and peroxisomal bifunctional enzyme with SB-219994, were identified in this manner.

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In the carnivorous dipteran Sarcophaga bullata Parker, vitellogenesis was partially inhibited by injection of two doses of 12 μg abscisic acid (ABA), There was no significant difference between injections on day 2 and 4, or on day 4 and 6. Higher and lower doses were less effective, The mixture of isomers inhibited vitellogenesis more than the cis‐trans isomer. ABA had no effect on the total lipid concentration of the haemolymph but it inhibited the sharp increase in total protein concentration of the haemolymph and particularly that of vitellogenin, which normally occurred within 24 hr following liver feeding on day 4, Since vitellogenin synthesis is under the control of moulting hormone (MH), the MH activity was measured by radioimmunoassay to find out whether ABA might interact with vitellogenin synthesis via its hormonal inductor. Eight hours after liver feeding, there was a MH peak in the control groups while following ABA treatment this peak occurred after 18 hr, The inhibitory effect of ABA on vitellogenesis could be overruled by feeding sugar impregnated with ecdysterone (5 mg/g), not by topical application of JH. Our results suggest that ABA might interact with a mechanism which phytophagous and non‐phytophagous insects share in common. If in phytophagous insects the same amount of ABA per gram weight, as was effective in Sarcophaga (about 200 μg/g), is needed to reduce fecundity, it is not probable that this plant hormone plays a role in the seasonal synchronisation of the growth and reproduction of insects with the senescence of their host plants. RÉSUMÉ EFFET DE L'ACIDE ABSISIQUE SUR LA VITELLOGENESE DE SARCOPHAGA BULLATA La vitellogenèse du diptère carnivore, Sarcophaga bullata a été partiellement inhibéc par injection de deux doses de 12 μg d'acide absisique (ABA). Il n'y avait pas de différence significative entre les injections du deuxième et quatrième jour, ou du quatrième et sixième jour La mixture d'isomères a plus inhibé la vitellogenèse que le cis‐trans isomère. ABA n'a pas eu d'effet sur la concentration totale des lipides de l'hémolymphe, mais il a inhibé l'augmentation brutale de la concentration protéique totale de l'hémolymphe, et particulièrement celle de la vitellogénins qui se produit normalement dans les 24 h après consommation de fore le 4ème jour La synthèse de la vitellogémne étant sous le contrôle de l'hormone de mue (MH), l'activité MH a été mesurée par radio‐immunologie, pour découvrir si l'ABA agissait sur la synthèse de la vitellogénine par intermédiaire de son inducteur hormonal. Huit heures après alimentation sur foie, le taux de MH chez les témoins était élevé tandis qu'après traitement avec ABA ce pic n'apparaissait qu'après 18 h. L'effet inhibiteur de l'ABA sur la vitellogénine a pu être neutralisé en fournissant aux mouches du sucre impregné d'ecdystérone (5mg/g), mais non par application locale d'hormone juvénile. Nos résultats suggèrent qu'ABA agit probablement sur un mécanisme commun aux insectes phytophages et non phytophages. Il est peu probable que l'acide absisiq...

No abstract

The considerable interest in the Group II secreted phospholipase A, (PLA,; EC 3.1.1.4) as a possible mediator of the inflammatory response is because of the association of elevated levels of this enzyme with inflammatory disorders as in septic shock patients.An important question in this area of inflammation research is the origin of this serum PLA,. In a previous communication, we have highlighted the liver as a possible source of this enzyme particularly as the activity of this enzyme was elevated in liver after pretreatment of animals with endotoxin (1).In a separate study by Besler and Grimble [2], an enhanced inflammatory response to endotoxin challenged in rats fed for 28 days on a 10% corn oil (polyunsaturated) diet was observed as compared to animals on an olive oil (monounsaturated) diet and in rats fed an equivalent saturated diet consisting of butter fat.In this communication, we have investigated the effect of endotoxin challenge on the activity of the liver Group II PLA, enzyme which is normally associated with the mitochondrial fraction from a rat liver homogenate [3] and, in particular, how this endotoxin challenge might be modulated by diet.Male Wistar rats (1 50 -2009 body wt), were used throughout the study. In the acute endotoxin response investigation, the rats were injected intraperitoneally with a dose of 10 mglkg of pure LPS from Escherichia coli K-235. After a 4 hour incubation, the rats were sacrificed. The liver was removed and homogenised using the same method as Hatch eta/ [l] to obtain the mitochondrial fraction. A celldebris and nuclei free supernatant was centrifuged at 105,000 g for 60 min to isolate a total membrane fraction. The pellets were resuspended in buffer B (0.25 M sucrose, 20 mM Tris/HCI, pH 7.4, 1 mM EDTA, 1 M KCI). The samples were assayed for PLA, [4]. These samples were also sonicated and centrifuged at 350.000 g for 15 min in a Beckman Optima TLX ultracentrifuge. The supernatants were then assayed for PLA, activity [4].In the 28 day diet study, rats were fed on a standard chow diet and nine experimental diets which varied in the type and level of fat. Corn oil, olive oil and butter were included in the diet at different concentrations -5%, 10% and 20% by weight. A single dose of 0.8 mg/kg of Escherichia coli endotoxin (DIFCO strain 055:B9) was administered interperitonally to half the rats. A similar dose of sterile saline was given to the control rats. After 24 hours the rats were sacrificed. The livers were removed and processed using the procedure essentially described above. One possible explanation for the endotoxin effect on enhancing PLA, activity [l] might be a redistribution and/or increased releasibility of PLA, activity. In order to address this problem the Table 1. The effect of endotoxin on PLA, activity in rat liver.Sample was assayed as a crude fraction without the final centrifugation step at 350.000 g for 15 min to remove the membrane Darticles after KCI treatment and sonication.