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INTRODUCTION
The neurotoxic potential of aluminium (Al) is generallyaccepted. Its causal role in dialysis-related encephalopa-thy (DE) is well recognized (Alfrey et al., 1976; Flen-drig et al., 1976; Alfrey & Froment, 1990). Its possiblerole in Alzheimer’s disease (AD) (McLachlan et al.,1992; Wisniewski & Wen, 1992), amyotrophic lateralsclerosis (ALS) and Parkinsonism-dementia (PD) of Guam (Garruto, 1991) is not yet unravelled. Al is capa-ble of inducing dementia-like symptoms and cytoskele-tal abnormalities involving neurofilaments andmicrotubular proteins in neurons
in vivo
in experimen-tal animals (Garruto et al., 1989; Garruto et al., 1991;Strong & Garruto, 1991b; Strong etal., 1991). Even
invitro
, using neuronal cell culture systems, cytoskeletaldamage and induction of neurofibrillary tangles areobserved (Langui et al., 1988, 1990; Roll et al., 1989;Shea et al., 1989; Van Welsum et al., 1989; Shi & Haug,1990; Hewitt et al., 1991; Strong & Garruto, 1991a).These experimentally induced phenomena are not com-pletely similar to those in humans.The role of fluoride in relation to Al neurotoxicity iscontroversial. When Al and fluoride are given togetherin the diet or in the drinking water not only protectiveeffects against neurotoxicity are reported (Still & Kel-ley, 1980; Forbes et al. 1991; Forbes & McAiney, 1992;Kraus & Forbes, 1992) but also neurotoxicity-enhancing effects (Isaacson et al., 1997; Varner et al.,1998). Also, systemic fluoride is reported to enhancethe systemic accumulation and toxicity of Al (Stevenset al., 1983; Isaacson et al., 1997). A specific Alfluoride compound, AlF
4–
, has been known for a longtime as a non-specific activator of guanine nucleotidebinding-proteins (G-proteins) and consequently thephosphoinositol (PI) transduction system (Bigay et al.,
Archives of Physiology and Biochemistry1381-3455/99/10701-0015$15.001999, Vol. 107, No. 1, pp. 15–21©Swets & Zeitlinger
F
LUORIDE
E
NHANCES THE
E
FFECT OF
A
LUMINIUM
C
HLORIDEON
I
NTERCONNECTIONS
B
ETWEEN
A
GGREGATES OF
H
IPPOCAMPAL
N
EURONS
G.B. van der Voet, O. Schijns and F.A. de Wolff 
Toxicology Laboratory, Leiden University Medical Center, Leiden, The Netherlands
ABSTRACT
The role of fluoride in aluminium neurotoxicity was studied using an in vitro system of cultured hippocampal neuronsfrom foetal rats. Sodium fluoride (50
M) and aluminium chloride (12.5
M) were administered alone or in a specificcombination (50
12.5
M) in a 14-day culture in a chemically defined medium before staining of neurofilaments.Neuronal aggregates interconnected by neuritic fibers were detected light microscopically in control cultures. The aggre-gates and the fibers stained positive for neurofilament proteins. In cultures treated with aluminium chloride the develop-ment of the interconnecting fibers was affected, resulting in a fusion pattern of the aggregates. This phenomenon wasenhanced when sodium fluoride was given together with aluminum chloride.It was concluded that aluminium interferes with the metabolism of the neuronal cytoskeleton and that this interfer-ence is potentiated by fluoride.KEYWORDS: Aluminium, fluoride, cell culture, aggregates, neurofilaments, cytoskeletal proteins, neurotoxicity.Address correspondence to: Dr. G.B. van der Voet, Toxicol-ogy Laboratory, Leiden University Medical Center, PO Box9600 – 2300 RC Leiden, (Albinusdreef 2 (building 1, floorL1) – 2333 ZA Leiden), The Netherlands. Phone
31 71 5262202; Fax
31 71 526 6759
 
1985, 1987). Therefore, Al fluoride may be, more thanAl chloride, expected to have an effect on the intra-cellular calcium status.It may be hypothetized that the speciation of Al fluo-ride is strongly predisposing for the passage routeschosen over the intestinal wall, blood-brain-barrier andcell membranes and the development of neurotoxicity.Therefore, to resolve the role of fluoride in Al neurotox-icity, cultured hippocampal neurons from foetal ratswere treated with a specific combination of Al chlorideand sodium fluoride (1:4) to establish a specific chemi-cal species to possibly stimulate the PI system and todetect any effect on cytoskeletal proteins, especiallyneurofilaments, and to compare these data with resultsfrom cultures treated with Al chloride. This
in vitro
sys-tem of cultured hippocampal neurons, growing as aggre-gates, using a chemically defined medium, allowed tocircumvent all physiological barriers (intestinal wall,blood-brain-barrier) as encountered
in vivo
and was con-sidered a better model for the brain than monolayer orneuroblastoma culture. Moreover, this system had beenproven useful to detect cytoskeletal effects of Al chloridein previous studies (Van Welsum et al., 1989)
MATERIALS AND METHODSCell cultures
Timed-pregnant Wistar-rats were anaesthetized withether and sacrificed by cervical dislocation. Theabdomen was opened and the 19-day-old embryos wereremoved from the uterus and rinsed in 70% ethanolduring maximal 15 to 20 minutes. In a sterile environ-ment the embryos were decapitated and the brainsremoved. After rinsing the brains in sterile 0.9% salinethe hippocampi were dissected and collected in thechemically defined medium R-12. The hippocampiwere mechanically dissociated with a flame-polishedPasteur pipette to obtain a cell suspension. The prepa-ration and composition of the chemically defined cul-ture medium and the dissociation procedure wereadapted from the earlier work by Romijn etal. (1984),Van Dorp et al. (1990) and Walsh et al. (1990). Thiscell suspension was washed twice in R-12 medium andthen centrifuged at 1000 rpm in a table centrifuge for 5minutes. The cells were resuspended in A) R-12medium (control), B) R-12 medium
NaF (50
M),C) R-12 medium
AlCl
3
.6H
2
O (12.5
M) and D) R-12 medium
AlCl
3
.6H
2
O (12.5
M)
NaF (50
M).These different cell suspensions were put on poly-D-lysine coated petri-dishes (poly-D-lysine: 0.01%,Sigma, St. Louis, USA, No. 2659). The cells wereallowed to attach to the petri-dishes for 3-4 hours in a37°C water-saturated atmosphere containing 5% CO
2
.Then 1 ml R-12 medium was added to the petri dishes.The medium was renewed for the first time after 24 hand then every subsequent 2 days. At this moment the14-day culture could be started. On all days from day 1to 14 eight cultures were terminated (four culturesduplex) by addition of Histofixative (Merck) after theculture medium was removed.
Staining procedures
The cultures were processed for the detection of neuro-filament (NF) proteins using monoclonal antibodiesagainst NF 90, which detects neurofilamental proteinsof 70, 150 and 200 kDa (Oudega et al., 1990). NF 90immunoglobulin is a monoclonal immunoglobulin pro-duced by a hybrid cell line from SP
2
O/Ag
14
myelomacells and lymphocytes from the spleen of a Balb/Cmouse. First the fixative was removed by rinsing twicewith phosphate-buffered saline (PBS)(0.1 M; pH
7.2)during 5 minutes, followed by rinsing once with PBS
0.1% bovine serum albumin (BSA) during 5 minutes.The first incubation with NF 90 took place overnight atroom temperature in a water saturated atmosphere. Theimmunoglobulin was diluted 1:10.000 in PBS
0.1%BSA
1% NSG (normal goat serum). Then the cul-tures were rinsed again twice with PBS for 5 minutesevery time and then each with PBS and 0.1% BSA oncefor 5 minutes. The second incubation, using rabbit anti-mouse Ig/peroxidase was performed 3–4 h in a solutionof PBS, 0.1 % BSA, NSG 1% at room temperature in awater-saturated atmosphere. Thereafter, the cultureswere rinsed again twice with PBS for 5 minutes. Justbefore the incubation 3 mM H
2
O
2
was added. Then thecultures were incubated with 80 mM 3, 3-diaminoben-zidine-4HCl (DAB) in 0.05 M Tris-maleïne buffer (pH
7.6) for 5–10 minutes. After incubation the DABsolution was removed and tap water was added, fol-lowed by rinsing with distilled water. The cultures werestained with hematoxyline-eosine, and dehydratedthrough graded alcohol to xylene and mounted in Depex(DPX)(Pearse, 1972). Finally, the cell cultures wereinspected using light microscopy.
RESULTSControls
Neuronal aggregates were observed in the control cul-tures (Fig. 1a). These aggregates appeared as solitary16
THEROLEOFFLUORIDEINALUMINIUMTOXICITY
 
G
.
B
.
VANDERVOETETAL
.17
Fig.1a.Aggregates of hippocampal neurons interconnected by neuritic fibers after a culture period of 6 days in R-12 medium (NF 90 staining,magnification
40, light microscopy).Fig.1b.Aggregates of hippocampal neurons with interconnecting fibers after a culture period of 6 days in R-12 medium containing 50
M NaF(NF 90 staining, magnification
40, light microscopy).
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