Effects of the solvent 1,2,4-trimethylcyclohexane on respiratory burst in human neutrophil granulocytes: A chemiluminescence and electron paramagnetic resonance spectrometry study

We investigated the effects of 1,2,4-trimethylcyclohexane (TMCH, 10-50 µM), n-nonane (10-50 µM) and 1,2,4-trimethylbenzene (TMB, 0.05-0.5 mM) on free radical production in vitro in human neutrophil granulocytes and rat brain synaptosomes. Human granulocytes and rat brain synaptosomes were incubated with 2',7'-dichlorofluorescein diacetate prior to exposure of the organic solvents. Reactive oxygen species (ROS) production was subsequently measured by chemoluminescence spectroscopy. The solvent-induced elevation of free radicals in human granulocytes were also measured by EPR spectroscopy after incubation with the spin trap DEPMPO. Mechanisms for ROS production was elucidated by use of enzymatic inhibitors. Low concentrations of TMCH, n-nonane and TMB stimulated respiratory burst (production of O2-) in human granulocytes and ROS production in rat brain synaptosomes. The respiratory burst in response to TMCH was dependent of extracellular calcium. U-73122, a phospholipase C (PLC) inhibitor, and 7,7-dimethyleicosadienoic acid, a phospholipase A2 (PLA2) inhibitor lowered the TMCH-stimulated O2- production in granulocytes. Furthermore, bisindolylmaleimide, a protein kinase C (PKC) inhibitor, and diphenyleneiodonium, a NADPH-oxidase inhibitor, reduced the TMCH-stimulated respiratory burst almost completely. In conclusion, these results indicates that TMCH-activated respiratory burst is dependent of Ca2+-dependent PLC, PLA2 and PKC prior to activation of the NADPH-oxidase.