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
O. Myhre et F. Fonnum, 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, BIOCH PHARM, 62(1), 2001, pp. 119-128
This study investigated the effects of C7 and C9 aliphatic (n-heptane, n-no
nane), naphthenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) an
d aromatic (toluene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on the prod
uction 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 produce
d a concentration-dependent linear increase in the formation of the fluores
cence of 2',7'-dichlorofluorescein (DCF) as a measure of the production of
ROS and RNS. Formation of RNS was demonstrated by preincubation of the syna
ptosome fraction with the neuronal nitric oxide synthase (nNOS) inhibitor N
omega -nitro-L-arginine methyl ester (L-NAME), which reduced the MeHg and
TMCH-stimulated fluorescence by 51% and 65%, respectively. The naphthenic h
ydrocarbon 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 intrace
llular calcium concentration [Ca2+](i) as measured with Fura-2. Blockade of
voltage-dependent Ca2+ channels with lanthanum prior to stimulation with M
eHg and TMCH led to a reduction in the ROS/RNS formation of 72% and 70%, re
spectively. 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 synapt
osome fraction with the protein tyrosine kinase inhibitor genistein lowered
the MeHg and TMCH-stimulated fluorescence by 85% and 91%, respectively. Ad
dition 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 r
educed the fluorescence stimulated by MeHg and TMCH by 52% and 56%. The com
pound 1-(6-[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]-aminohexyl)-1H-pyr
role-2,5-dione (U73122), which inhibits phospholipase C, was shown to decre
ase the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, resp
ectively. The phospholipase A(2) (PLA(2)) inhibitor 7,7-dimethyl eicosadien
oic acid (DEDA) reduced fluorescence in response to MeHg and TMCH by 49% an
d 54%. Simultaneous addition of L-NAME, CSA, and DEDA to the synaptosome fr
action totally abolished the DCF fluorescence. In conclusion, C7 and C9 ali
phatic, naphthenic, and aromatic hydrocarbons stimulated formation of ROS a
nd RNS in rat brain synaptosomes. The naphthenic hydrocarbon TMCH stimulate
d formation of ROS and RNS in the synaptosomes through Ca2+-dependent activ
ation of PLA(2) and nNOS, and through increased transition permeability of
the MTP. Exposure of humans to the naphthenic hydrocarbon TMCH may stimulat
e formation of free radicals in the brain, which may be a key factor leadin
g to neurotoxicity. (C) 2001 Elsevier Science Inc. All rights reserved.