The effect of aliphatic, naphthenic, and aromatic hydrocarbons on production of reactive oxygen species and reactive nitrogen species in rat brain synaptosome fraction: the involvement of calcium, nitric oxide synthase, mitochondria, and phospholipase A11Abbreviations: BIM, bisindolylmaleimide; [Ca2+]i, concentration of intracellular calcium; ChAT, cholin acetyltransferase; CSA, cyclosporin A; DCF, 2′,7′-dichlorofluorescein; H2DCF-DA, 2′,7′-dichlorodihydrofluorescin diacetate; DEDA, dimethyleicosadienoic acid; ERK, extracellular signal-regulated kinases; Fura-2 AM, 5-oxazolecarboxylic acid, 2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(bis(2-((acetyloxy)methoxy)-2oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benzofuranyl)-, (acetyloxy) methyl ester; GABA-T, gamma-aminobutyric acid transaminase; HBSS, Hanks’ balanced salt solution; La3+, lanthanum; MAPK, mitogen-activated protein kinase; MeHg, methyl mercury; MEK, extracellular signal-regulated protein kinase; MeOH, methanol; MTP, mitochondrial permeability transition pore; L-NAME, Nω-nitro-l-arginine methyl ester; NO·, nitrogen oxide; NOS, NO· synthase; O2·−, superoxide; PLA2, phospholipase A2; PKC, protein kinase C; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; TMB, 1,2,4-trimethylbenzene; TMCH, 1,2,4-trimethylcyclohexane; and U73122, 1-(6-[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-aminohexyl)- 1H-pyrrole-2,5-dione

Autor: Oddvar Myhre, Frode Fonnum
Rok vydání: 2001
Předmět:
Zdroj: Biochemical Pharmacology. 62:119-128
ISSN: 0006-2952
Popis: This study investigated the effects of C7 and C9 aliphatic (n-heptane, n-nonane), naphthenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) and aromatic (toluene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat brain synaptosome fraction. Methyl mercury (MeHg) was included as a positive control. Exposure of the synaptosomes to the hydrocarbons produced a concentration-dependent linear increase in the formation of the fluorescence of 2',7'-dichlorofluorescein (DCF) as a measure of the production of ROS and RNS. Formation of RNS was demonstrated by preincubation of the synaptosome fraction with the neuronal nitric oxide synthase (nNOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME), which reduced the MeHg and TMCH-stimulated fluorescence by 51% and 65%, respectively. The naphthenic hydrocarbon TMCH showed the strongest potential for ROS and RNS formation in rat brain synaptosomes, followed by TMB, toluene, n-nonane, n-heptane, and methylcyclohexane, respectively. TMCH was selected for mechanistic studies of the formation of ROS. Both MeHg and TMCH induced an increase in intracellular calcium concentration [Ca(2+)]i as measured with Fura-2. Blockade of voltage-dependent Ca(2+) channels with lanthanum prior to stimulation with MeHg and TMCH led to a reduction in the ROS/RNS formation of 72% and 70%, respectively. Furthermore, addition of cyclosporin A (CSA), a blocker of the mitochondrial permeability transition pore (MTP), lowered both the MeHg and TMCH-elevated DCF fluorescence by 72% and 59%. Preincubation of the synaptosome fraction with the protein tyrosine kinase inhibitor genistein lowered the MeHg and TMCH-stimulated fluorescence by 85% and 91%, respectively. Addition of the extracellular signal-regulated protein kinase (MEK)-1 and -2 inhibitor U0126 reduced the fluorescence stimulated by MeHg and TMCH by 62% and 63%. Furthermore, the protein kinase C inhibitor bisindolylmaleimide reduced the fluorescence stimulated by MeHg and TMCH by 52% and 56%. The compound 1-(6-[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122), which inhibits phospholipase C, was shown to decrease the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, respectively. The phospholipase A2 (PLA2) inhibitor 7,7-dimethyl eicosadienoic acid (DEDA) reduced fluorescence in response to MeHg and TMCH by 49% and 54%. Simultaneous addition of L-NAME, CSA, and DEDA to the synaptosome fraction totally abolished the DCF fluorescence. In conclusion, C7 and C9 aliphatic, naphthenic, and aromatic hydrocarbons stimulated formation of ROS and RNS in rat brain synaptosomes. The naphthenic hydrocarbon TMCH stimulated formation of ROS and RNS in the synaptosomes through Ca(2+)-dependent activation of PLA2 and nNOS, and through increased transition permeability of the MTP. Exposure of humans to the naphthenic hydrocarbon TMCH may stimulate formation of free radicals in the brain, which may be a key factor leading to neurotoxicity.
Databáze: OpenAIRE
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