TOXICOLOGY AND APPLIED PHARMACOLOGY 57, 8- 13 ( 1981)

Effects of Thallium, Nickel, and Cobalt Administration on the Lipid Peroxidation in Different Regions of the Rat Brain

MAHDI HASAN AND S. FATEHYAB ALI

Brain Research Center, Jawaharlal Nehru Medical College, Aligarh Muslim University, ALIGARH-202001, India

Received May 5. 1980; accepted August 25. 1980

Effect of Thallium, Nickel, and Cobalt Administration on the Lipid Peroxidation in Dif- ferent Regions of the Rat Brain. HASAN. M., AND ALI. S. F. (1980). Tosi~l. Appl. Phrrrr~- c,ol. 57, 8-13. Rats were given thallium (5 mg/kg), nickel, and cobalt (2 mg/kg) ip daily for 7 days. Clinically, thallium induced a maximum degree of toxicosis, followed by nickel and cobalt. When determined after 7 days of treatment, the rate of lipid peroxidation was significantly increased in the cerebrum, cerebellum, and brain stem of rats in all treatment groups. Thallium caused a maximum increase in the rate of lipid peroxidation in the cere- bellum but cobalt and nickel produced maximum effects in the brain stem.‘Electron micros- copy revealed increased lipofuscin-like pigment deposition in the cerebellar neurons fol- lowing thallium-intoxication. This correlated well with the increased rate of lipid peroxidation.

Peroxidation involves the direct reaction of oxygen and lipid to form free radical inter- mediates and semistable peroxides. Lipid peroxidation is damaging because of the subsequent reactions of free radicals, mainly peroxy radicals, that are produced (Tappel, 1970). Lipid peroxidation in vivo

has been claimed to be of basic importance in aging, in damage to cells by air pollution and in oxygen toxicity (Tappel, 1973). Quantitative studies of enzyme inactivation by lipid peroxidation have shown that sulf- hydryl enzymes are most susceptible to in- activation (Chio and Tappel, 1969). Thal- lium is known to combine with sulfhydryl groups, resulting in a deficiency of cysteine leading secondarily to a deficiency of glu- tathione (Gross et al., 1948). Furthermore, a decrease of total sulfhydryl groups in mitochondria causes their swelling and de- generation. Since thallium intoxication is known to induce mitochondrial changes (Hasan et al., 1977, 1978), we decided to

test the effect of thallium on the rate of lipid peroxidation and to correlate it with electron microscopic observations for lipofuscin deposition. Interestingly, Sesame and Boyd (1978) have reported paradoxical effects of cobalt chloride and salts of other divalent metals (including nickel) on tissue levels of reduced glutathione. Because there is a re- lationship between glutathione and lipid peroxidation (Tappel, 1970), one aim of the present study was to evaluate the effects of nickel and cobalt on the rate of lipid peroxidation in different regions of the rat brain.

METHODS

Male albino rats of the Charles Foster strain weighing 150 c 20 g, were fed a commerical diet (Hind Lever Laboratory Feeds, India). There were 10 treated rats in each of three treatment groups and 10 control rats. They were housed in a group of not more than four in plastic cages with dimensions of 12 x 9 x 6in.

0041-008x/81/010008-06$02.00/0 Copyright ‘D 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.

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LIPID PEROXIDATION IN RAT BRAIN 9

Biochemical Determinations

Treatment. The solution of thallous acetate, nickel chloride, and cobalt chloride (BDH, Poole, Dorset, England) were prepared in double-distilled water. Rats of group 1 were injected with a thallous acetate solution ip (5 mg of thallium/kg) daily for 7 days. Rats in groups 2 and 3 were given nickel chloride solution (2 mg of nickel/kg) and cobalt chloride solution (2 mg of cobalt/kg) ip daily for 7 days. Rats in group 4 served as controls and were given appropriate volumes of physiological saline ip concurrently.

Lipid peroxidation. Rats were fasted overnight (allowing water ad lihitum) and sacrificed by de- capitation. Brains were dissected into cerebral hemisphere, cerebellum. and brain stem in a cold room and weighed to the nearest milligram on an electri- cal balance. Brain from the respective areas was homogenized in chilled 0.15 M potassium chloride in a Potter-Elvehjem type C homogenizer (Arthur H. Thomas & Co., Philadelphia, Pa.) with a Teflon pestle, and the final volume adjusted to have a 10% (w/v) homogenate. For assay of lipid peroxidation, 1.0 ml of homogenate was incubated at 37 2 1°C in a metabolic shaker (120 strokesimin; amplitude 1 cm) for 3 hr. One milliliter of 10% (w/v) trichloroacetic acid was added and after thorough mixing, the reaction mixture was centrifuged at 800 g for 10 min. Samples (1 ml) of the clear supematant were mixed with 1 ml of 0.67% (w/v) 2-thiobarbituric acid and held in a boiling water bath for 10 min, cooled, and diluted with 1 ml distilled water. The absorbance of the solution was read at 535 nm and the results expressed as nanomoles of malonaldehyde formed per 30 min using the extinction coefficient 1.56 x IO5 as described by Utley et ul. (1967).

Electron microscopp. Twelve male albino rats of the same strain were divided into two groups of six each. Rats in Group I were given thallous acetate solution ip (5 mg thallium/kg) daily for 7 days and equal volumes of physiological saline were con- currently given ip to rats in Group 2. The brains of all the rats were fixed by perfusion fixation with phosphate buffered glutaraldehyde/parafonnaldehyde solutions. prepared along the lines recommended by Karnovsky (1965). Two rats were anesthetized at a time (one experimental and one control) with ip sodium pentobarbital (30 mg/kg body wt) and per- fused through the heart. The perfusion fluid was kept at a height of about 100 cm above the level of the heart of the animal. the needle was introduced into the left ventricle and guided into the ascending aorta. The duration of perfusion was 25-30 min and the volume for each animal was approximately 300 ml (at the rate of about 10 mllmin). The brains were removed from the cranium, taking care to avoid trauma. Small pieces of the cerebellum were dis- sected out and quickly immersed in the fixative used

for perfusion for 3 hr at 4°C. The tissue was then rinsed in 0.1 M phosphate buffer (pH 7.3) and postfixed in 1% osmotically adjusted osmium tetroxide for l-2 hr. After dehydration the material was embedded in a mixture of araldite 502 and Epon 812 (1:2). The de- sired regions of the brain were sectioned ( 1 pm thick I with an LKB Ultratome Model III and stained with toluidine blue. Adjacent ultrathin sections were ob- tained from the area of interest. They were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined with a Hitachi IIU HE electron microscope at an accelerating voltage of 75 kV.

The data were analyzed statistically using Student’s t test. Significant differences between the means of the experimental and control groups were calculated and p values were obtained.

RESULTS

General Observations

Signs of thallium toxicosis included anorexia, failure to gain weight, poor hair luster, lethargy, diarrhea, dragging of hind- limbs, and fits of abnormal rotation of head and neck. Treatment with nickel and cobalt caused irritability and restlessness. After 4-5 days all the treated rats usually became lethargic. Control rats remained normal.

Necropsy

Congestion of meninges and signs of minimal cerebral oedema were observed at necropsy in some rats in all the treatment groups. The weight of brains from thallium, nickel, and cobalt treated rats, however, did not differ significantly from control values. Coronal sections of the brain at various levels did not reveal gross ab- normality. The ventricular system was bi- laterally symmetrical.

Biochemical Findings

Lipid peroxidation. The rate of lipid peroxidation, expressed as nanomoles of

10 HASAN AND ALI

TABLE 1

RATE OF LIPID PEROXIDATION EXPRESSED AS NANOMOLES OF MALONALDEHYDE FORMED PER 30 min IN

DIFFERENT REGIONS OF THE RAT BRAIN AFTER THE ip ADMINISTRATION OF THALLIUM (5 mgikg) FOR 7 DAYS (MEAN 2 SE)

Parts of brain

Cerebral hemisphere Cerebellum Brain stem

Control (N = 10)

2.482 i_ 0.333 2.305 c 0.319 1.430 + 0.173

Experimental (thallium) (N = 10)

3.701 -c 0.317 5.584 + 0.448 3.100 rt 0.350

Percentage change

+49 +142 +116

P

<O.OOl <O.ool <O.ool

Nore. N = Number of animals used in each experiment.

malonaldehyde formed per 30 min in dif- ferent regions of the brain of control and thallium-intoxicated rats are shown in Table 1. Significant increases in the rate of lipid peroxidation were maximal in the cerebel- lum followed by brain stem and cerebral hemisphere. Values for the rate of lipid peroxidation after the administration of nickel and cobalt are presented in Table 2. There was a significant increase in peroxi- dation in the cerebellum and brain stem in cobalt toxicosis but in the cerebral hemi- sphere the change was insignificant. In the case of nickel toxicosis, the rate of lipid peroxidation was markedly increased in all three regions of the brain in the same pattern we found in thallium intoxication.

Electron microscopy. Cerebellar neurons of the control rats showed well-preserved endoplasmic reticulum, mitochondria, and

glogi zones (Fig. 1). Lipofuscin pigment granules, however, were scarcely dis- cernible. On the other hand, prominent aggregation of lipofuscin granules were frequently observed in the perikarya of cerebellar neurons obtained from thallium- intoxicated rats (Fig. 2).

DISCUSSION

No comparable report on alterations in the rate of lipid peroxidation following the administration of thallium, cobalt, or nickel is available. The amount of malonicdialde- hyde produced, as measured by the thio- barbituric acid (TBA) assay, has been shown to be a true indicator of endoge- nous lipid peroxidation (Tappel and Zalkin, 1960). The brain homogenate apparently has the necessary unsaturated fatty acids and

TABLE 2

RATE OF LIPID PEROXIDATION EXPRESSED AS NANOMOLES OF MALONALDEHYDE FORMED PER 30 min IN DIFFERENT REGIONS OF THE RAT BRAIN AFTER THE ADMINISTRATION OF COBALT AND NICKEL (2 mgikg body wt ip DAILY FOR 7 DAYS) (MEAN t SE)

Experimental Per- Experimental Per- Control (cobalt) centage (nickel) centage

Parts of brain (N = 10) (N = 10) change P (N = 10) change P

Cerebral hemisphere 2.482 _t 0.333 2.454 ? 0.301 -2 N.S. 3.327 + 0.321 +34 co.01

Cerebellum 2.305 _t 0.319 3.095 r 0.261 +34 <O.Ol 3.442 c 0.331 +40 <O.oOl Brain stem 1.430 t 0.173 2.596 2 0.251 +80 <O.OOl 2.217 k 0.221 +55 ~0.001

Note. N = Number of animals used in each experiment.

LIPID PEROXIDATION IN RAT BRAIN

FIG. 1. Electron micrograph of a part of the cerebellar cortex of the control rat. Note the remark- able paucity of the lipofuscin granules. The cytoplasmic organelles are well preserved. x 16,000 (scale == I pm).

the catalysts for peroxidation in the archi- dation product to lipofuscin has been tecture of the cell itself which are readily verified on the basis that it gives fluores- available for reaction with molecular oxygen cence products which have characteristic to undergo lipid peroxidation. Biomem- fluorescence spectra with a maximum at 470 branes and subcellular organelles are the nm when excited at 365 nm, similar to those major cellular components damaged by lipid of lipofuscin pigment (Chio et ~1.. 1969). peroxidation. At least two in viva systems The increased incidence of electron dense protect against membrane damage resulting bodies observed in cases of thallium toxi- from uncontrolled lipid peroxidation. These cosis appear to be the end result of lipid systems rely on selenium and vitamin E peroxidation. Also, cobalt chloride and salts and form the basis for hypotheses concern- of other divalent metals (including nickel) ing the antioxidant functions of these are known to alter tissue levels of reduced nutrients (Combs et al., 1975). Thallium glutathione (Sesame and Boyd, 1978). Since combines with sulfhydryl groups resulting the association of glutathione with lipid ultimately in a deficiency of glutathione peroxidation is well recognized (Tappel, (Gross rf nl., 1948). This causes deficient 1970) the increased rate of lipid peroxi- degradation of lipid peroxides to hydroxy dation following cobalt and nickel adminis- acids leading to the accumulation of the tration can be easily explained. One of the former in various regions of the brain most interesting observations, however. is (Tappel, 1970). The similarity of the peroxi- the regional heterogeneity of the percentage

12 HASAN AND AL1

FIG. 2. Electron micrograph of thallium (5 mgikg)-treated rat. Three polymorphic lipofuscin granules are visualized in the perikaryon of a cerebellar neuron. Note electron lucid vacuole in one lipofuscin pigment (arrow). X14,400 (scale = I Fm).

increase in the rate of lipid peroxidation in the various regions of the brain caused by different metal ions. Whereas thallium caused maximum increase in the rate of lipid peroxidation in the cerebellum, cobalt, and nickel produced maximum effect on the brain stem. On the other hand, cobalt did not significantly alter the rate of lipid peroxi- dation in the cerebrum but nickel did cause a 34% increase in this region as well. A certain selectivity of cells, terminals and enzymes to the toxic effets of cobalt in viva has been reported by Clayton and Emson (1976).

multineuronal pathways in the brain stem. However, no direct cot-relationship between the signs of their toxicity with the charac- teristics regional differences in lipid peroxi- dation could be established.

ACKNOWLEDGMENTS

The authors are grateful to Dr. A. C. Shipstone, Incharge, Electron Microscope Division of C.D.R.I., Lucknow for some facilities provided for this work. Thanks are also due to the Council of Scientific and Industrial Research, New Delhi, India, for awarding a Research Fellowship to one of us (S. Fatehyab Ali).

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