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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