Effect of alcohol intake on some disturbances induced by chronic exposure to carbon disulphide in rats, II. Biochemical and ultrastructural alterations in the peripheral nerves

Toxicology Letters, 24 (1985) 171-177

171

Elsevier

TOXLett.

1357

EFFECT OF ALCOHOL INTAKE ON SOME DISTURBANCES INDUCED BY CHRONIC EXPOSURE TO CARBON DISULPHIDE IN RATS, II. BIOCHEMICAL AND ULTRASTRUCTURAL ALTERATIONS IN THE PERIPHERAL NERVES (Carbon

disulphide;

JOLANTA

OPACKA,

ethanol;

peripheral

TERESA

neuropathy;

WRONSKA-NOFER,

rat)

JAN

KOLAKOWSKI*

and

BARBARA

OPALSKA*

Department of Biochemistry and *Department of Pathomorphology, Medicine, Teresy 8, 90-950 Lodz, P.O. Box 199 (Poland) (Received

October

(Accepted

December

26th,

Institute

of Occupational

1984)

21st,

1984)

SUMMARY The effect of chronic studied.

ethanol

Rats were exposed

the last 4 months peripheral

of the experiment.

nerve were estimated

ed. It was shown that EtOH nerves,

(EtOH)

i.e. an increase

ratio) and a decrease

on CSz-induced

Some biochemical

and ultrastructural

augmented

in cholesterol

the alterations

correlates

examinations

peripheral

content.

of Wallerian

of the peripheral

in lipid content

The magnitude

neuropathy

and to 10% EtOH

in rats was

as only drink

during

degeneration

of the

nerves were perform-

provoked

esters and in the ratio of cholesterol

in phospholipid

in the nerves was increased

administration

to 0.8 mg/l CS2 for 12-15 months

by CS2 in the peripheral

esters to free cholesterol

of ultrastructural

changes

induced

(E/F by CSz

by EtOH.

INTRODUCTION A problem of growing interest is the combined effect of EtOH and industrial solvents. So far very little is known about the case of EtOH and carbon disulphide [l], particularly with regard to the nervous system in experimental animals, although it is well known that both CS2 and EtOH are neurotoxic. In our previous study [2] we found that combined exposure to CS2 and EtOH might affect the function of the central nervous system (learning and memory in rats were depressed). Abbreviations:

EtOH,

ethanol;

E/F,

ratio of cholesterol

esters to free cholesterol;

vous system.

0378-4274/85/S

03.30 0 Elsevier

Science

Publishers

B.V.

PNS, peripheral

ner-

172

In the present paper we studied the effect of EtOH on CSz-induced alterations in the peripheral nervous system (PNS) of rats. It has been documented in morphological studies [3,4] that chronic exposure of rats to CS2 produces peripheral neuropathy characterized by the Wallerian-type axonal degeneration in which damage to the axon precedes disintegration of the myelin sheet. The nature of the peripheral neuropathy caused by chronic EtOH intake is a matter for discussion. Some data suggest a primary injury of the axon [5], others myelin degradation [6]. In our former study [7] we found that some biochemical correlates of Wallerian degeneration - i.e. the activity of fl-glucuronidase and the ratio E/F - were affected in C&induced axonopathy, and as the magnitude of these changes was associated with the degree of exposure, they could be useful as biochemical indicators of C&-induced neuropathy. In the present work, the biochemical measurements were supplemented by ultrastructural observations of peripheral nerves of rats. We found that EtOH augmented the disturbances in lipid content provoked by CS2, as well as the ultrastructural changes in the PNS. MATERIALS

AND

METHODS

Female white rats of the Wistar strain inhaled CSZ vapour at a concentration of 0.8 mg/l for 5 h per day, 6 days per week for 12 or 15 months. During the last 4 months of exposure to CS2 the rats were given only 10% EtOH to drink. Water and alcohol consumption was controlled daily. Intake of EtOH during the 4 months of exposure was 7-10 g/kg/day in both ‘EtOH’ and ‘CS2 + EtOH’ groups, without significant differences between those groups, and independent of the exposure period. The control rats were treated and kept in chambers under the same conditions as the exposed animals, without CS2 intoxication. 20 h After the last daily exposure to CS2, the animals were killed by decapitation. After bleeding, the sciatic and tibia1 nerves were isolated. For biochemical evaluation, nerves were frozen immediately in liquid nitrogen, and used for cholesterol, phospholipid and pglucuronidase determination according to the methods described previously [7]. For ultrastructural examination, nerves were fixed in 3,6% glutaraldehyde, postfixed in 1% osmium tetraoxide, processed through increasing concentrations of EtOH and embedded in Epon 812. Semithin sections stained with toluidine blue were prepared for preliminary examination by light microscopy. Thin sections were examined after staining in uranyl acetate and lead citrate, with a JEM 100-C electron microscope. RESULTS

A long-term (12-15 months) inhalation of CSZ vapour evoked in the rat PNS an increase of /3-glucuronidase activity and some disturbances in lipid content (Fig. l), principally an increase in esterified cholesterol and in the E/F ratio. The content of total and free cholesterol, as well as that of phospholipids, was slightly decreased.

173 (A)

200 * 180 160 140. 120 ’ 100. 80.

I

,

T

F

E

E/F

PL

8-GLC

T

F

E

EIF

pL* B-GLG

Fig. 1. Biochemical alterations in the peripheral nerves of rats exposed to EtOH ez.rzq CSZ n and CS2 + EtOH ~cmm,expressed as % control. Mean control values I SD.: total cholesterol (T), 26.0 + 5.1 mg/g tissue; cholesterol esters (E), 1.5 k 0.7 mg/g tissue; E/F ratio, 0.061 + 0.034; phospholipids (PL) as lipid phosphorus, 2.5 -t 0.3 mg/g tissue; @-glucuronidase @GLC), 1.79 & 0.35 pmol/h/g tissue. Number of’ animals per group: 7-10; (A) Rats exposed to 0.8 mg CSz/l air for 12 months and to 10% ethanol in drinking water for the last 4 months of the experiment, (B) Rats exposed to 0.8 mg CSJI air for 1.5months and to 10% ethanol in drinking water for the last 4 months of the experiment. *f’
These changes were more pronounced after 15 months of exposure to CS2. Administration of EtOH only did not significantly affect the above parameters. But EtOH as an additional toxic factor elevated the Iipid alterations induced by CS2; mostly an increase in cholesterol esters and in the E/F ratio as well as, to a lesser extent, a decrease in phospholipids was observed. No effect of EtOH on the changes in ,&glucuronidase activity provoked by CS2 was observed. Biochemical alterations were accompanied by changes in the morphological structure of axons. 4 months’ consumption of 10% EtOH resulted in mild changes in a few axons, in which a decreased number of neurotubules was found. Morphological alterations in myelin fibres of the peripheral nerves of rats exposed to CS2 for 12 months were related to axons and Schwann cells. Increased numbers of neurofilaments, and disappearance of neurotubules in the axoplasm were observed (Fig.2). Multivesicular structures and vacuoles loaded with glycogen were also found in some myelin fibres. Enlargement of rough endoplasmic reticulum canals, activation of Golgi apparatus and large amounts of free ribosomes in the Schwann cells were observed. Injured fibres were phagocytosed by active Schwann cells. The

Fig. 2. The sciatic

nerve of a rat exposed

neurofilaments

(F). Sparse neurotubules

magnification.

Normal

Fig. 3. Semithin

proportions

cross-section

to 0.8 mg C&/l

(T) are seen.

of neurofilaments

of the sciatic

x

air for 12 months.

16600.

Inset: Control

Axoplasm

filled with

sciatic nerve in the same

and neurotubules.

nerve of a rat exposed

to 0.8 mg C&/I

air for 12 months

(A) and that of a rat exposed to 0.8 mg CSz/l air for 12 months and to 10% ethanol in drinking water for the last 4 months of the experiment (B) Increased numbers of enlarged axons are seen in (B) compared to (A).

x 800.

175

morphological alterations in the myelin fibres of CSz-exposed rats after EtOH administration were similar to those induced by CS2 alone, but were found in a greater number of fibres (Fig. 3, A and B). Degenerated fibres were observed, ons were filled with neurofilaments and vesicles of various diameters

in which ax(Fig. 4).

DISCUSSION

C&-induced peripheral neuropathy, classified as filamentous [8], is morphologically characterized by swelling of the axon and accumulation of neurofilaments [3,4]. The biochemical alterations found in this axonopathy in rats are demonstrated by an increase in cholesterol esters, in the E/F ratio and in the activity of P-glucuronidase [7]. In the present paper it was shown that the alterations induced by CS2 in the rat PNS, both biochemical and ultrastructural, were augmented by chronic administration of EtOH. EtOH affected some biochemical correlates of C&-induced neuropathy, mainly the lipid parameters: cholesterol ester content, E/F ratio and, to a lesser extent, phospholipid content. The activity of P-glucuronidase was unaffected by EtOH. All parameters tested in the present study were the correlates of

Fig. 4. The sciatic nerve of a rat exposed to 0.8 mg C&/l ing water for the last 4 months of the experiment. various shapes and sizes. x 16600.

Axon

air for 12 months

and to 10% ethanol

filled with vesicular

and myelin

in drink-

structures

of

176

a second stage of peripheral nerve degeneration, i.e. cell proliferation and chemical degradation of myelin lipids [9]. The ultrastructural changes found in the peripheral nerves of rats exposed to C’S2 and EtOH were qualitatively similar to those observed after CS2 alone. EtOH increased the magnitude of C&-provoked changes which are typical of the axonopathy. No signs of demyelinisation of the nerve fibres were found. These findings indicate that chronic EtOH intake augmented the axon damage induced by CS2. The problem of pathogenesis of combined exposure to CS2 and EtOH on the PNS is interesting. Alcoholic polyneuropathy was often understood to be a consequence of B-vitamin deficiency [ 10,111. The toxic effects of CS2 were also considered to be a result of disturbances in the metabolism of B-vitamins [12,13]. It is possible that the increase in CS2 neurotoxicity due to chronic EtOH administration results from a disturbed balance in vitamin metabolism; this hypothesis should be tested experimentally.

ACKNOWLEDGEMENTS

The authors wish to thank Mrs. Wiesiawa Adamiak and Mrs. Zofia Rudnicka for their technical help. This work was supported by the Polish-American Agreement No. 05-008-2 and by Agreement No. Br. 8.01.76 with the Polish Artificial Fibres Union.

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