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 A
Oddvar Myhre
a,b,
*, Frode Fonnum
a,b
a
Norwegian Defence Research Establishment, Division for Protection and Material, P.O. Box 25, N-2027 Kjeller, Norway
b
VISTA (The Norwegian Academy of Science and Letters/Statoil)
Received 1 August 2000; accepted 16 January 2001
Abstract
This study investigated the effects of C7 and C9 aliphatic ( n-heptane, n-nonane), naphthenic (methylcyclohexane, 1,2,4-trimethylcy-
clohexane (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 N-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 phospho-
lipase C, was shown to decrease the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, respectively. The
phospholipase A
2
(PLA
2
) 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
PLA
2
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. © 2001 Elsevier Science Inc. All
rights reserved.
Keywords: Reactive oxygen species; Reactive nitrogen species; Aliphatic, naphthenic, and aromatic hydrocarbons; Rat brain synaptosomes
Biochemical Pharmacology 62 (2001) 119 –128
0006-2952/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0006-2952(01)00652-9