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