Toxicology
Letters,
213
51 (1990)213-219
Elsevier
TOXLET
02307
Effects of aluminum on brain lipid peroxidation
Cesar G. Fraga l**, Patricia I. 0teiza2,*, Mari S. Golub3, M. Eric Gershwin3 and Carl L. Keen2,3 ‘Department
of Biochemistry,
University of California. Berkeley,
of California, Davis, CA and3Department California, Davis, CA (U.S.A.) (Received
18 August
(Accepted
2 November
CA, 2Department
of Nutrition,
of Internal Medicine, School of Medicine,
University
University of
1989) 1989)
Key words; Brain; Lipid peroxidation;
Aluminum
toxicity
SUMMARY Excessive dietary disorders taining
(Al) has been proposed
Six I-week-old
100 (control,
was determined brain
aluminum
in humans.
by evaluating
the production in the presence
of iron in brain homogenates
the 100 Al control
group
TBARS production
of 2-thiobarbituric or absence
TBARS
for the 3 groups.
production production
protein).
suggesting
in the presence
diets con-
and absence measured
neurological
damage
associated
with increased
in in
(30%) than that in iron increased
TBARS production
(4.9 vs. 3.9 nmol TBARS/mg
of iron was similar between either with or without
Al intoxication
lipid peroxidation
(TBARS) production
of ferrous
The iron-induced
than in the 100 Al group
in liver homogenates
that enhanced
substances
iron. TBARS
The addition
from all 3 dietary groups.
These results show that, in mice, dietary
production,
acid reactive
of 50 PM ferrous
(3.1 vs. 2.4 nmol TBARS/mg
in brain homogenates
Brain TBARS
500 Al groups.
to several neurological
from mice fed the 1000 Al diet was higher
was 26% higher in the 1000 Al brain homogenates protein).
contributing
mice were fed for 10 weeks purified
100 Al), 500 (500 Al) or 1000 (1000 Al) pg Al/g diet. Brain and liver lipid peroxidation
and liver homogenates
the absence
to be a factor
female Swiss Webster
leads to increased
may be one possible
the 100 and
iron was similar
mechanism
brain TBARS underlying
the
tissue Al.
INTRODUCTION
Aluminum (Al) is the most abundant metal in the earth’s crust, but until recently it was considered to be of low toxicological significance. At neutral pH, Al exists mainly as Al(OH)3 which, due to its high insolubility, results in low levels of Al in *Current address: Departamento
de Quimica
Biologica,
Facultad
de Farmacia
y Bioquimica,
Universidad
de Buenos Aires, Junin 956, (1113) Buenos Aires, Argentina. Address for correspondence:
Dr. Carl L. Keen, Department
of Nutrition,
University
CA 95616, U.S.A.
0378-4274/90/S
3.50 @ 1990 Elscvier Science Publishers
B.V. (Biomedical
Division)
of California,
Davis,
714
surface waters. However, during the last decade it has been recognized that acid precipitation can markedly increase the mobilization of Al into surface waters [19]. In patients with chronic renal failure, Al in dialysis fluids has been shown to be an etiological factor for the encephalopathy, osteomalacic osteodystrophy and anemia previously associated with long-term dialysis treatment [21]. In individuals with normal renal function, Al has been proposed to be a factor contributing to several neurological disorders. Brain Al concentration may increase with age in humans [12] and Al is found in high concentrations in hippocampal neurons containing neurofibrillary tangles in Alzheimer’s disease [ 151, amyotrophic lateral sclerosis and parkinsonian dementia, as well as in neurologically ‘normal’ controls [16]. A recent study has shown that the incidence of Alzheimer’s disease was higher in districts with a high Al content in the drinking water than in low Al regions [ 131. The peroxidation of lipids and other cell components, e.g. proteins and nucleic acids, has been related to a number of pathophysiological situations [I, 10, 171. Aluminum has been shown to modify the extent of lipid peroxidation induced by exogenous iron in liposomes, red blood cell ghosts and liver microsomes [2, 9, 181. For these reasons we tested the hypothesis that dietary Al intoxication could enhance tissue lipid peroxidation. Mice were fed for 10 weeks either control diets (100 pg Al/g) or high Al diets (500 and 1000 pg Al/g) and the peroxidazibility of brain and liver homogenates was evaluated by measuring the formation of thiobarbituric acid reactive substances (TBARS). Consistent with our hypothesis, the peroxidazibility of the brain was increased in the 1000 pg Al/g group compared to the controls. However, liver TBARS production was not increased by dietary Al intake, showing that there is tissue specificity with regard to the effect of Al on lipid peroxidation. MATERIALS
AND METHODS
Atlimuls Female Swiss Webster mice N:NIH(s) at 6 8 weeks of age were purchased from a commercial source (Matsunaga Tayman Scientific, San Diego, CA) and randomly assigned to one of the 3 treatment groups. Mice were fed ad libitum purified diets prepared commercially (Dyets, Inc., Bethlehem, PA) in pelleted form. which contained 100 (control: 100 Al). 500 (500 Al) or 1000 (1000 Al) pug Al/g diet, as Al lactate, for 10 weeks. The control level of dietary Al was chosen based on the analysis of commercially available mouse chows, which typically range from 25 to 100 /lg Al/g. The 1000 Al diet used in this study has been shown to produce a low-level toxicity syndrome when fed for 6 weeks [8].
Mice were euthanized by decapitation and brains and livers were quickly dissected, rinsed and homogenized in Krebs-Ringer phosphate buffer (pH 7.4). To assay tissue peroxidazibility, aliquots of 0.5 ml of homogenate (about 50 mg of fresh tissue) were
215
incubated ferrous
at 37°C sulfate.
with continuous
The reaction
agitation,
was stopped
in the presence
by placing
or absence
the samples
of 50 ,uM
at 4°C and adding
0.1 ml of 4% (w/v) butylated hydroxytoluene in ethanol to prevent further peroxidation. The determination of TBARS was performed fluorimetrically as described by Fraga et al. [7]. The values were expressed as nanomoles of TBARS (malondialdehyde equivalents) per mg protein. The effect of added Al was determined by incubating from mice fed control diets in the presence of Al2(SO&
brain and liver homogenates in the incubation medium.
Aluminum analysis Determination of Al concentration in liver was carried out as previously described [ 141 using a graphite furnace atomic absorption spectrophotometer with a super-pressure mercury lamp. Protein determination Protein concentration was determined vine serum albumin as standard. Statistical analysis One-way analysis
as described
by Lowry et al. [I l] using bo-
of variance
routines available in Statview compared by Fisher’s method
and regression analysis were performed using the 512+ (Brainpower Inc., Calabazas, CA). Means were of protecting least significant differences.
Time (min) Fig. 1, Time-course
production
and (B) liver homogenates iron (50 PM) for different
Time (min)
of TBARS
were incubated time periods.
in mouse brain and liver homogenates. at 37”C, in the absence
(0)
The conditions used are described section.
(A) Brain homogenates
or the presence in the ‘Materials
(0)
of ferrous
and Methods’
RESULTS
Animal outcome and Al levels Consistent with previous observations. body weight gain over the IO-week period was similar among the experimental groups [14]. Liver and brain weights, both on an absolute basis and on a body weight basis, were similar among the groups. To evaluate the accumulation of Al in the tissues, liver concentrations of the metal were determined. Liver Al in the 1000 Al group (3.0 + 0.9 pmol/g wet wt.) was significantly higher (PC 0.05) than in the 100 Al (0.6 + 0.1 pmol/g) and 500 Al (I .O f 0.3 pmol/g) groups. Al on TBARS,formation by brain and liver homogenates In both the presence and absence of 50 mM FeS04, the production of TBARS by the liver and brain homogenates increased with the incubation time (Fig. IA and B). An incubation time of 15 min brought an adequate estimation of the peroxidazibility of the tissue. TBARS production by brain homogenates was higher than that of liver homogenates. Table I shows the results obtained from mice fed diets containing different amounts of Al. Brain homogenate TBARS production in the absence of added ferrous sulfate was correlated with the amount of Al present in the diet (r =0.97. P < 0.05). In the group fed the 1000 Al diet the production of TBARS in brain homogenates was significantly higher (P-C 0.05, 30%) than in the 100 Al group. Increasing amounts of Al in the diet also enhanced the production of TBARS in brain homogenates incubated in the presence of 50 PM ferrous sulfate (r = 0.95, P < 0.05) and the l$ect
cfdietary
TABLE
I
EFFECT
OF DIETARY
ALUMINUM
ON TEARS
PRODUCTION
IN LIVER
AND
BRAIN
HO-
MOGENATES Diet
TBARS (nmolimg
protein)
Brain
Liver
No additions
+Fe
No additions
+Fe
100 Al
2.4 kO.2.1
3.9,0.3,’
0.71 kO.06
1.4iO.2
500 Al
2.5kO.2
4.3 50.3
0.74kO.06
1.5*0.2
1000 Al
3.1 kO.lh
4.9i0.6h
0.68 +0.06
1.3*0.1
Tissue homogenates
were incubated
for
I.5min at 37”C, either in the absence or presence of 50 PM ferrous
sulfate. After incubation, TBARS were analyzed fluorimetrically as described in ‘Materials and Methods’. Data are presented as mean f SEM and are the average of 6 animals per group; (a) significantly different from (b). P
217
TABLE
II
EFFECT
OF ALUMINUM
ADDITION
ON TBARS PRODUCTION
BY LIVER
AND BRAIN
HO-
MOGENATES Aluminum
TBARS
01M)
(nmol/mg
protein) Liver
Brain No additions
0.62 f 0.03
5.8kO.3
0.46kO.04
1.7*0.1
10.5+0.6
0.52kO.06
3.5kO.l
were incubated
for 15 min at 37°C
50 PM FeSO+ After incubation,
and Methods’.
2.1*0.1
5.4*0.5
Tissue homogenates and/or
+Fe
1.00+0.05 0.72kO.04
1000
No additions
0.78 f 0.03
0 100
+Fe
Data are shown as means
TBARS
either in the absence
were analyzed
fluorimetrically
or presence
of Alr(SO&
as described
in ‘Materials
+ SEM (n = 3).
peroxidazibility of brain homogenates from mice fed the 1000 Al diet was 26% higher than that of the 100 Al group (PcO.05). In contrast to brain, the production of TBARS by liver homogenates was similar for the 3 dietary groups (Table I). Effect of Al addition on TBARSproduction by brain and liver homogenates The effect of direct Al addition to brain and liver homogenates obtained from control animals is shown in Table II. In the absence of added iron, Al diminished the formation of TBARS with a maximum effect at 100 ,uM. This antioxidant-like effect was reverted by higher Al concentrations in the presence of 50 ,uM FeS04, the addition of 100 ,LLMAl did not modify TBARS production, but 1000 ,uM Al increased TBARS production by 94 and 67% over control samples in brain and liver homogenates, respectively. DISCUSSION
Results from this study show that high levels of dietary Al fed to adult mice for 10 weeks increased the peroxidazibility of brain homogenates. In contrast to the brain, the peroxidazibility of liver homogenates was not modified by dietary Al feeding. The peroxidazibility of tissues is an evaluation of the presence of substrates for lipid peroxidation reactions (polyunsaturated fatty acids, metals and 02) and of antioxidants (vitamin E, vitamin C, glutathione, antioxidant enzymes), and its quantitation (in this case, the presence of TBARS) reflects the result of oxidative reactions occurring in vivo [20]. Thus, the differential effect on brain and liver peroxidazibility observed in the animals fed high dietary Al may be a consequence of a higher rate of in vivo production of oxidative reactions occurring in the brain.
21x
The effects of metals
on free radical
reactions
is usually
ascribed
to their ability
to participate in redox reactions, in which they donate or accept a single electron. Al, due to its electronic configuration, does not participate in redox reactions. Consequently, the effect of Al is probably due to a direct interaction with cell components, rather than due to reactions with oxidative radical species. For example, Al binds to negatively charged vesicles with a higher affinity than that of Ca2+ [4] and, as a result, can affect the physical state of membranes [5, 61. Thus the differential effect of Al on brain versus liver peroxidazibility may be related to differences in membrane lipid composition and/or surface charge density; a higher binding of Al to the membrane would be predicted to cause a greater rearrangement of the membrane phospholipids, which should render the lipids more accessible to the attack of free radicals. When Al was added to brain homogenates, we observed a dual effect of the metal: an antioxidant-like effect at physiological concentrations of iron, and a pro-oxidant effect at higher iron concentrations. We suggest that, in the absence of added iron, the antioxidant effect of Al is related to its ability to bind to membranes and displace membrane-bound iron. thus preventing iron-catalyzed oxidative reactions. In contrast, high Al and Fe concentrations increased the formation of TBARS over control values. This oxidant effect is probably due to a disorganizing effect of Al on the membranes together with a high iron availability. On the other hand, this oxidant effect of Al is not tissue-specific, suggesting that the in vivo effect of Al on brain is related to an accumulation of Al in the brain membranes. It is important to note that in phosphate buffers the free A13+ concentration is determined by the solubility of AlP04.2HlO. At pH 7.4, 0.16 mol/l ionic strength [3] and in the presence of 10 mM phosphate concentration, the maximum free Al’+ concentration can be estimated to be 1.9 x lOpI4 M. Thus, under the conditions used in the present study, the vast majority of the Al is either membrane-bound or complexed with inorganic phosphate. In summary, our results suggest that the diseases of the central nervous system associated with the presence of Al may have free-radical-mediated oxidative reactions as causative or propagating mechanisms. ACKNOWLEDGEMENTS
Supported in part by NIH grant GM 04190. CGF has a fellowship from Consejo National de Investigaciones Cientificas y Tecnicas de la Republica Argentina. We want to thank Prof. A.L. Tappel, in whose laboratory CGF did part of this work. REFERENCES 1 Ames, B.N. (1983) Dietary carcinogens eases. Science 22 1, 1256 1264.
and anticarcinogens:
oxygen
radicals
and degenerative
dis-
219
2 Aruoma,
O.I., Halliwell,
nism of initiation plex. Biochem. 3 Martin,
B., Laughton,
M.J., Quinlan,
of lipid peroxidation:
G.J. and Gutteridge,
evidence against
J.M.C. (1989) The mecha-
for an iron (IIkiron (III) com-
a requirement
J. 258,617620.
R.B. (1986) The chemistry
of aluminum
as related
to biology
and medicine.
Clin. Chem. 32,
179771806. 4 Deleers,
M. (1985) Cationic
branes:
pathological
atmosphere
implications
and cation
competition
of aluminum.
Res.
binding
Commun.
at negatively
Chem.
charged
Pathol.
mem-
Pharmacol.
49,
277-294. 5 Deleers, M., Servais, J.P. and Wiilfert, aration,
aggregation
E. (1985) Micromolar
Acta 813, 1955200. 6 Deleers, M., Servais, J.P. and Wiilfert, and membrane 7 Fraga,
fusion at micromolar
C.G., Leibovitz,
somes. Free Radical MS.,
dation
J.M.C.,
10 Halliwell,
J.M., Gershwin,
Rosebrough,
Folin phenol reagent. 12 Markesbery, 13 Martyn,
C.N.,
aluminum
W.D., Alauddin,
D.J.P.,
ingestion
on
salts accelerate
peroxi-
Acta 835,441447. FASEB J. 1,3588364.
R.J. (1951) Protein
measurement
with the
M. and Hossain,
Osmond,
C., Harris,
in mice. Toxicol.
con-
R.F. (1989)
water. Lancet
1, 5962.
evidence of aluminum
ac-
Science 208,297-299.
R.M., Yanagihara, lateral
of high
Lett. 47,279-285.
disease: X-ray spectrometric
neurons.
in amyotrophic
J.A. and Lacey,
in drinking
J.M. and Keen, C.L. (1989) The influence
polymerization
tangle-bearing
D.C., Garruto,
E.C., Edwardson,
disease and aluminum
M.E., Donald,
on brain microtubule
accumulation
T.I.M. (1984) Brain traceelement
Aging 5, 19-28.
Gershwin,
in neurofibrillary
al aluminum
Biophys.
disease: some new concepts.
between Alzheimer’s MS.,
16 Perl, D.P., Gajdusek,
B. (1985) Aluminum
A.L. and Randall,
15 Perl, D.P. and Brody, A.R. (1980) Alzheimer’s cumulation
and micro-
J. Biol. Chem. 193,2655275.
Barker, relation
14 Oteiza, P.I., Golub, dietary
as thiobarbituric
with homogenates
11,231-235.
by iron salts. Biochim.
and human
in aging. Neurobiol.
Geographical
Teratol.
G.J., Clark, I. and Halliwell,
N.J., Farr,
W.R., Ehmann,
centrations
measured
and comparison
M.E. and Keen, C.L. (1989) Effects of aluminum
lipids stimulated
B. (1987) Oxidants O.H.,
rigidification
Acta 855,271-276.
A.L. (1988) Lipid peroxidation
of mice. Neurotoxicol.
Quinlan,
of membrane
1 I Lowry,
induce membrane
Biophys.
Biophys.
Biol. Med. 4, 1555161.
motor activity
9 Gutteridge,
of Al)+ induce phase sep-
lipid vesicles. Biochim.
cations
Biochim.
in tissue slices: characterization
Donald,
spontaneous
E. (1985) Neurotoxic
concentrations.
B.E. and Tappel,
acid reactive substances 8 Golub,
concentrations
and dye release in phosphatidylserine-containing
R.T. and Gibbs, Jr., C.J. (1982) Intraneuron-
sclerosis
and Parkinsonian-dementia
of Guam.
Science 217, 1053~1055. 17 Pryor,
W.A. (1986) Oxy-radicals
and related
species: their formation,
lifetimes,
and reactions.
Annu.
Rev. Physiol. 48,657667. 18 Quinlan, aluminum crosomal 19 Sharpe,
G.J., Halhwell, fraction.
Biochim.
C.P. and Gutteridge, lipid peroxidation
Biophys.
20 Tappel, A.L., Tappel, tion processes.
of drinking
A. and Fraga,
J.M.C.
(1988) Action
of lead (II) and
erythrocytes
and rat liver mi-
in hposomes,
Acta 962, 196200.
W.E. and De Walle, D.R. (1985) Potential
and metals contamination
health implications
water, Environ.
for acid precipitation,
Health Perspect.
C.G. (1989) Application
corrosion
63,71-78.
of simulating
modeling
to lipid peroxida-
Free Rad. Biol. Med. 7, 361-368.
21 Wills, M.R. and Savory, Environ.
B., Moorhouse,
(III) ions on iron-stimulated
Health Perspect.
J. (1985) Water content 63, 141-147.
of aluminum,
dialysis,
dementia,
and osteomalacia.