(+/-)-2-Chloropropionic acid elevates reactive oxygen species formation in human neutrophil granulocytes.
(+/-)-2-Chloropropionic acid (2-CPA) is a neurotoxic compound which kills cerebellar granule cells in vivo, and makes cerebellar granule cells in vitro produce reactive oxygen species (ROS). We have studied the effect of 2-CPA on ROS formation in human neutrophil granulocytes in vitro. We found an increased formation of ROS after 2-CPA exposure using three different methods; the fluorescent probe DCFH-DA and the chemiluminescent probes lucigenin and luminol. Four different inhibitors of ROS formation were tested on the cells in combination with 2-CPA to characterize the signalling pathways. The spin-trap s-PBN, the ERK1/2 inhibitor U0126 and the antioxidant Vitamin E inhibited the 2-CPA-induced ROS formation completely, while the mitochondrial transition permeability pore blocker cyclosporine A inhibited the ROS formation partly. We also found that 2-CPA induced an increased nitric oxide production in the cells by using the Griess reagent. The level of reduced glutathione, measured with the DTNB assay, was decreased after exposure to high concentrations of 2-CPA. Western blotting analysis showed that 2-CPA exposure led to an elevated phosphorylation of ERK MAP kinase. This phosphorylation was inhibited by U0126. Based on these experiments it seems like the mechanisms for 2-CPA induced toxicity involves ROS formation and is similar in neutrophil granulocytes as earlier shown in cerebellar granule cells. This also implies that 2-CPA may be immunotoxic.
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Norwegian Defence Research Establishment, Division for Protection, P.O. Box 25, N-2027 Kjeller, Norway. bba@ffi.no <bba@ffi.no>
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D000166:Acridines; D015153:Blotting, Western; D002469:Cell Separation; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005978:Glutathione / Q000378:metabolism; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006843:Hydrocarbons, Chlorinated; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D049449:Luminescence; D008165:Luminol; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D011422:Propionates / Q000037:antagonists & inhibitors / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013439:Sulfhydryl Reagents; D015854:Up-Regulation / Q000187:drug effects
D000166:Acridines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006843:Hydrocarbons, Chlorinated; D007202:Indicators and Reagents; D009566:Nitrates; D009573:Nitrites; D011422:Propionates; D017382:Reactive Oxygen Species; D013439:Sulfhydryl Reagents; C029569:diacetyldichlorofluorescein; C033472:10,10'-dimethyl-9,9'-biacridinium; D008165:Luminol; D004228:Dithionitrobenzoic Acid; C021974:2-chloropropionic acid; D007770:L-Lactate Dehydrogenase; D048049:Extracellular Signal-Regulated MAP Kinases; D005978:Glutathione