Effects of aluminum on brain lipid peroxidation

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.

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