title: Pro-inflammatory effects of commercial alpha-lactalbumin on RAW 264.7 macrophages is due to endotoxin contamination.
abstract: This study investigated the effects of alpha-lactalbumin (alpha-LA) on cellular signaling molecules associated with inflammatory responses in RAW 264.7 macrophages. The results indicated that commercial alpha-LA could increase prostaglandin E(2) (PGE(2)) and the expression of COX-2 via increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and jun N-terminal kinase (JNK), and increase nitric oxide (NO) and the expression of iNOS via the activation of ERK1/2 and JNK. Furthermore, commercial alpha-LA could increase nuclear translocation of p65 nuclear factor-kappa B (p65 NF-kappaB) through stimulation on inhibitor kappa B-alpha (IkappaB-alpha) degradation. Since endotoxin also has these effects, we assayed the content of endotoxin in the commercial alpha-LA. We found to our surprise that endotoxin was there and that alpha-LA-induced NO and PGE(2) production could be suppressed by polymyxin B, a specific inhibitor of endotoxin. Thus, the pro-inflammatory effects of commercial alpha-LA might be caused by endotoxin contamination through activation and expression of iNOS and COX-2 which were upregulated by MAPKs or nuclear translocation of p65 NF-kappaB in RAW 264.7 cells. It is therefore crucial to assess the possibility of endotoxin contamination within any biological product being studied for immune augmenting activities before a meaning result can be obtained.
authors.affiliation: Laboratory of Biophysics, Department of Research and Education, Taipei Veterans General Hospital, Taipei 112, Taiwan.
authors.forename: I-Chun
authors.initials: IC
authors.lastname: Lin
authors.forename: Cheng-Deng
authors.initials: CD
authors.lastname: Kuo
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D051546:Cyclooxygenase 2 / Q000096:biosynthesis; D015232:Dinoprostone / Q000096:biosynthesis; D004340:Drug Contamination; D004731:Endotoxins / Q000032:analysis / Q000633:toxicity*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D007155:Immunologic Factors / Q000494:pharmacology; D007249:Inflammation / Q000139:chemically induced*; D007768:Lactalbumin / Q000032:analysis / Q000633:toxicity*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D009578:Nitrobenzenes / Q000494:pharmacology; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D004731:Endotoxins; D004791:Enzyme Inhibitors; D007155:Immunologic Factors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009578:Nitrobenzenes; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D009569:Nitric Oxide; D007768:Lactalbumin; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051546:Cyclooxygenase 2; D020928:Mitogen-Activated Protein Kinases; D015232:Dinoprostone; D019331:NG-Nitroarginine Methyl Ester