Cerebral changes in the course of intoxication with mercury phenylacetate

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Medical Academy Poznan , Poland Aut onomous Department of Pathology of th e Nervous System and of Sensory Organs (Head: Doc. Dr. hab. med. )1. B. KOZIK )

Cerebral changes in the course of intoxication with mercury phenylacetate With 10 figures (Received Januar y 19, 1978)

By M. B. KOZIK and J . WIGOWSKA -SOWINSKA Address: Doc. Dr. hab. med. )1. R. KOZIK, 49, Przybyszewskiego Str., 60 - 355 Poznan (Poland).

Key words: brain; central nervous system; nerve cells; cerebrum: intoxication; pesticides; fungicides ; mercury phenyla cetate: toxicity; nerve cells; cerebral phosphatases; cerebral esterases j rat

Sttmmary A histoenzymic study of cerebral phosphatases and esterases was performed on rats subjected to experimental intoxi cation with mercury phenylacetate. Following intragastric application of mercury phenylacetate to experiment al animals, decreased activities of cerebral ATPase, acP and AChE were observed. The into xicated animals displayed enhanced cerebral TPPase and partially also NsChE activities. Apart from changes in the histoenzymic pattern of the experimental brains, th e ingestion of mercury phenylacetate brought about evident morphological changes in form of neuronal vacnolisation and spongious degeneration of the white matter. The extent of morphological as well as histoenzymic alterations was dependent on the duration of the experimental poisoning. Mercury phenyl acetate is a pesticide with fungicidal properties. It has found application in agriculture (a s a seed mordant) in wood manufacture as well as in electrochemical industry (RUSIE CKI 1973). Like alkyl mer cury compounds, mercury phenylacetate possesses considerable toxicit y in man and animals. The particularly high vulnerability of the CNS has been frequently reported (CAVANAGH 1969; CHAXG and HARTMANN 1972). However, histochemical changes resulting in the brain from poisoning with this fungicide have not yet gained enough attention in studies dealing with aspects of toxicity of mercury compounds in the brain (SLIZEWSKI 1975; C HANG et al. 1972). The present study wa s undertaken in order to evaluate morphological and histochemical changes in the brain of experimental animals intoxicated subacutely and chronically with mercury phenylacetate.

111aterial and methods Three groups of Wistar rats of either sex, 150-180 g of body weight were used in the experiments. Group I, consisting of 14 rats received intragastrically mercury phenylacetate in single daily doses of 0.1 g each, during 10 consecutive days. Group II , consisting of 12 rats was fed intragastrically with 0.05 g of mercury phenylacetate during 30 consecutive days. The contr ol, untreated group consisted of 8 age matched, healthy animals. The experimental and control animals were killed under ethyl ether anaesthesia and their brains subjected to morphological and histoenzymic investigation. The brains were fixed in Baker's solution for 16 hrs at 4 °C. The fixed material was either embedded in paraffine or cut in a freezing microtome. Freely floating slices were subjected to appropriate histochemical reactions for demonstraing of activities of the following hydrolases:

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T'h i a m i n e p v ro p h n s p h at a s e I'I'PPasp) IE.L. ~.;i.l.3.l) - was assayed according to the method of ;'\()\'IKOFF and COLIlF1SCHEH (191)1 l, incubation conditions: room temp., 30 min. Nonspeeifie esterase (NsE) (KL. :eLl.1.1.) according to the method of NACHLAS and SELIGMANN (1949), incubation conditions: 37°C, 16 min. Ac e t y l c h o l i n e s t er as e (AChE) (E.I.. iLl.1.7.) - according to the method of GEREBTZOFF (1953), incubation conditions: :n DC, 1~0 min. No n s p e c if i c c h c l i n e s t e ra s e (NsChE) (E.L. i3.1.1.8.) - according to the method of GERETBZOFF (1953), incubation eonditions: 37°C, 180 min. Alkaline phosphatase (alkP) (KL. 3.1.3.1.) -- according to the method of GOMORI (1953), incubation time 30 min., temp. 37°C. Add phosphatase (acP) (E.L. :3.1.3.~.)- according to the method of GOMORI (1953), incubation time 60 min., temp. :i7 "C. Adenosine triphosphatase (A'l'Pase) (E.L. 3.5.1.3.) - according to the method of WACHSTEIN and MEISEL (1957), incubation time 45 min., temp. 37 DC. The paraffin brain slices were stained with cresyl violet, haematoxylin and eosine, and by the method of KLtVER-HAHHf:HA (19?iil).

Results Morphological changes First experimental group (subacute intoxication) In preparations from the subacutely intoxicated animals, there may be seen a widespread vacuolisation of neurocytes. This change occurred principally in the basal ganglia, in the anterior nucleus of the thalamus and in pyramidal cells of the Ammon's horn. Some of the thalamic nerve cells displayed signs of the chronic Nissl's disease and some others showed typical ischemic ehanges (ischemic necrosis]. Contrary to the basal nuclei, the cortex of the cerebral hemispheres lacked any notable neuronal changes. The white matter, when stained by the method of Kluver-Barrera showed multiple, small, spherical lacunae, presenting a

Fig. 1. Fornix. Widespread spongious changes with normal stainability of myelin sheaths. KluverBarrera staining. X 100.

1) E.L. ---- number of enzyme list (FLORKIN and STOTZ 1973).

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Fig. 2. Cortex of Ammon's horn, field CAL Besides neuronal vacuolisation, evident shrinkage of some neurocytes. Cresyl violet. x 900. condit ion kn own under t he te rm of spongious degenerati on (fig. I ]. There was no app ar ent demyelination det ect abl e in th e myelin of these experimental animals. Second experimen t al gr oup (chron ic intoxicat ion) Cyt oplasmic vaeuolisatio n of neur ons of man y cerebra l st ructures was t he out st anding morph ological change in t his experiment al group. Th e vac uolar degeneration was seen not only in th e anterior nu clei of t he thalamus, but also in t he medial and lateral ones. ~Iany neurons of th e striat um, th e globus pallidus and of the nu clei of cra nial nerves were similarly affecte d. In the cerebra l cortex and in the Amm on's horn , foca l neuronal shrinkage (fig. 2) as well as ischemic changes were seen. Th e cerebellum suffered frag ment ar y losses of Purkinj e cells and many cells displayed homogenisation changes. Thiamine pyrophosph at a s e (TP P ase) Contr ol group In normal conditions, th e histo chemical staining account ing for TPPase was localized pref erentially in blo od vessel walls and in the neur onal cyto plasm. The enzym e r eacti on pr odu ct s formed granular Of lamellar depo sits marking t he Golgi apparatus. Th e r eacti on was usually st ronger in the large pyramidal cells t han in t he gra nular zones. Th e oligo- and microglia of norm al rats was virtua lly unreacti ve. In some anima ls, single proto plas matic astroglial cells displaying a weak staining for TPPase were seen. Fi rst experiment al grou p Anima ls fed mercur y ph enylacetate during 10 consecutive days demonstrate d enha nced TPf'ase acti vit y in some ne urocytes, particularly in t hose forming th e Vt h cortical layer, in pyramidal cells of th e Ammon's horn and in neur ons residin g in the fascia dentat a (fig. 3). In addition to that a considerab le number of oligodend rocytes of the corpus callosum and

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of th e optic tract hecarne reactive with respect tn T PPase. Unlike control rat s, th e SlI Uacutely into xicated a nimals doveloped 'I'Pl'ase activity in the Bergmann 's oligcdendroglia of t he cerebellar cortex.

Fig. 3. Ammon's horn cortex. Rats tr eated with mercury phenylacetate for 10 days . The pyramidal and granular cells demonst rate considerable increase of TPPase activity. X 65. Fig. 4. Ammon's horn cortex. Very st rong TPPase activity in animals intoxicated with mercury ph enylacetat e for 30 days. X 65.

Fig. 5. Globus pallid us. Control group. Distin ct AChE activity in th e neuropil and neuro cyt es. >:: 160. Fig. 6. Globus pallidus. IInd experimental group. In comparison with contr ol preparations, evidently decreased AChE activi t y. x 160.

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Second experimental group The long-term ingestion of mercury phenylacetate resulted in a further and considerable increase of TPPase activity in the pyramidal cells of the cerebral cortex, in neurons of the Ammon's horn (fig. 4) and in those of basal ganglia and the cerebellum. The oligodendroglia of the cerebral and eerebellar white matter became also distinctly reactive. Nonspecific esterase (NsE) Control group The large pyramidal cells as well as the neuropil of the cerebral cortex showed appreciable reactivity for NsE. The fibrocytes of the soft meninges, the choroid plexus as well.as pericytes were strongly reactive. In the basal nuclei, there was distinct enzyme activity only in the large nerve cells. Moderate enzyme activity was also demonstrable in the pyramidal cells of the Ammon's horn as well as in neurons of the dentate fascia. The Purkinje cells of the cerebellum were appreciably reactive, whereas those of the granular layer showed considerably less NsE activity. First experimental group Like in control brains, distinct NsE activity was demonstrable in all larger neurocytes and in perieytes. Only the Purkinje cells of the subacutely intoxicated rats showed sometimes decreased enzyme activity, Second experimental group The localisation and intensity of the histoenzymic reaction for NsE in the chronically intoxicated animals did not differ from that observed in control animals. Acetylcholinesterase (AChE) Control group In the cerebral cortex, AChE activity was localised principally in capillary walls and in single "hyperactive" large neurocytes. The individual basal nuclei differed very much with respect to AChE activity. In some of them, a positive reaction was seen only within the perikaryons, in others, both the perikaryons and the neuropil were reactive whereas in still others, it was only the neuropil which exhibited AChE activity (fig. 5). First experimental group Ingestion of mercury phenylacetate for 10 days caused a slight decrease of AChE activity in the neuropil of the cerebral cortex and basal ganglia. The cortical perikaryons, like those of the cerebellar cortex, the pons and oblongate medulla remained unaffected with respect to AChE. Second experimental group The long-term administration of the poison brought about a moderate decline of AChE activity in the neuropil of the Ammon's horn cortex, of the basal nuclei, the pons and oblongate medulla, the decrease in enzymic activity being most pronounced within the globus pallidus (fig. 6). Nonspecific cholinesterase (NsChE) Control group In the cerebral and cerebellar cortex and white matter NsChE activity was seen only in blood vessel walls (fig. 7), whereas in the dorsal parts of the medial and lateral nuclei of the thalamus, there were also many neurocytes, positively stained for NsChE. First experimental group The histoenzymic reactivity for NsChE of the brain of animals treated with mercury phenylacetate for 10 days remained unaffected.

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7 Fig. 7. Temporal lobe. Control group. Moderate NsChE activit y ill capillar y walls. X 160. Fig. 8. Temporal lobe. Iln d experiment al group. Thickened capillar y walls and increased NsChE activity. X 160.

Second experiment al group Unlike in cont rol br ains or in th ose from th e first experiment al group, in brains of chronically intoxicat ed rats, t he ca pillary walls were thickened and th eir NsChEactivit y modera tely increased (fig. 8). Al ka l i ne ph osph a t a s e (alkP) Cont rol animals P yramidal cells of t he Ammon's horn , Purkinj e cells, neurocyt es of th e lateral part of th e rhin encephalon as well as t he nuclei of the V and VII cra nial nerves showed a weak , diffusive staining for alkP . Th e capillaries of the whole bra in were weakl y reacti ve as well. First experiment al gro up Th e localizati on and intensi ty of alkP in th e brain of subac utely int oxicated rats remained essentially un chan ged. Second experimental group Long-term administrat ion of mercur y phenylacet at e did n ot affect ' the histo enzymi c pattern of cerebral alkP. A cid phosphatase (aeP) Control group Cortical neurons as well as neur ocytes of the basal ganglia were reactive with respect t o acP . Th e acti vit y was located intracytoplasmatically, th e hist oenzymic reaction pr odu cts formin g granules of vari ous sizes and /or irr egularly sha ped dep osits. In the cerebellar corte x, distinct aeP activity was exhibite d only byP urkinje cells, whereas in the granular and molecular layers th e reaction was only weak and diffusive. F irst experimenta l group When compared wit h cont rol brains, the cort ical neur ocyt es of animals t rea te d with mercury phenyla eet at e for 10 days showed a generalized decrease of acP activity. Considerabl y lower activit ies were seen only in the cort ex of th e Ammon's horn, particularly within field CAL

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Fig. 9. Septum lucidum. Control group. ATPase activity in blood vessel walls and in perivascular processes of astrocytes. X 160. Fig. 10. Septnm lucidum. lInd experimental group. Perivascular feet of astrocytes lacking ATPase activity, and capillary walls displaying considerably reduced staining for ATPase. X 160. Second experimental group The long-t erm appl icat ion of t he poison caused a further decline of acP in most cortical n eurocyt es, A marked decrease of enzyme acti vit y is seen also in th e basal ganglia and in t he Purkinj e cells. Cont rar y t o t hat , th e nu clei of cra nial nerves retain ed normal acP activity. A d e n os i n e t r ip h osp hat a s e (ATPase) Cont rol group All blood vessels demonstrat ed distin ct ATP ase activit y and thi s both in the cerebra l corte x, in t he ba sal ganglia and in t he cerebellum. In t he Ammon's horn , in sept um lucidum as well as in t he basal ganglia th ere was in add iti on to vessel walls, considerable ATPase activit y demonst rab le in th e perivascular pr ocesses of prot oplasmati c astrocytes (fig. 9). F irst experimental group Animals t rea te d for 10 days with mercur y ph enylacet at e showed a clear-cut dr op of ATPase activity in capillari es of many cerebral regions, and thi s mainly in th e thalamu s, sept um lucidum, globus pallidus and striat um. In th ese regions, a distinct, positive hist oenzymic reaction for ATPase was seen only in prec apillari es and small arteries. Second experimental group In animals chroni cally int oxicated with mercury ph enylacetat e, ATPase activity of man y capillaries decreased subst antially or even disapp eared entirely (fig. 10). The cereb ra l cortex present ed vast areas, where ATPa se acti vity was defined only to larger blood vessels, t he capillary walls being complete ly unr eacti ve.

Discussion The ext ensive vacuolisation of nerv e cells as well as t he ap peara nce in the whit e matter of mul tipl e small cavities , a ph enomenon known und er t he term of' spongious or micr ocystic degeneration are the dominating cellular changes found in the brains of rats intoxicated experimentally with mercur y phenylacetate. 18 Exp. Path. Bd. 16

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Neuronal va cuolisati on is a Irequeutl y observed res ponse to acut e intoxication by va rious noxiou s agents, inst ead , spongious degeneration of th e white matter evolve s only exce pti onall y following th e action of toxic subst ances. Ult rast ruc t ural st udies of ESCOu ROLLE and POIRIER(l971) sugges t t hat microcysts , which are responsible for what is called th e spo ngious degeneration , develop insid e ast rocy te s. However, t he path ogencti c mechani sm leading t o the formation of th ese int racellular cav it ies is so far not ent irely dear. Besid es dismetab olic, degenerative or circulato ry concept s. assumptions are ra ised as t o a viral etiopat hogenesis of th ese cha nges, in spite of th e tact that th ey ar e not accompani ed by inflamm at ory sy mpt oms. Th e results of our experiments ha ve shown that spongious changes ma y in fa ct develop also in "t he course of some kind s of poisoning, as for inst an ce in th e cour se of mer cur y ph enylacetate intoxication. Our observ at ion does, of course , not explain the pathogenesis of thi s event, it does, however , exte nd th e list of etiolo gical fact ors capable of inducing spongious degeneration in the brain and is t herefore worth pointin g out. Th e histo enzymi c alte ra t ions brought about by mercur y phenylac etate poisonin g deserv e separat e consideration. Among th e inve stigated hydrolyt ic enzymes of th e brain acP , ATPase and AChE suffered a considerable decrease of act ivit y, TPPase inst ead was the only enzyme showing enha nced activity following into xicati on by mercury phenyla cetate, Prolonged, chronic administration of the poison over a period of 30 days, caused signifi cant augmentation of the effect s 0 bserv ed in animals ingesting mercury phenylacetate for 10 days only. Similar effects on th e act ivit y of cerebral hydrolases ha ve been observed in exp erim ental poisoning with corro siv e sublimat e and calomel (KOZIK et al. 1977 a and b). How ever, the ext ent and intensity of hist oenzymi c effects exerted hy mercur y ph enylacetate was obvi ously sma ller than tho se indu ced b y t he inorganic mer cur y salt s. Our observat ions ar e thus not in agr eement with those mad e by MIYAKAWA and DESHIM ARU (1968), CAVANAGHI(HI63) and CHANG et al. (1968) according to which organic mercur y compounds ar e stronger poinsons for the CNS than inorgani c ones. Thi s comm only accepted view thus seems t o need reconsidera t ion.

Conclus£ons 1. Intoxication of rats with mercur y phenylacetate brings about morphological cha nges in the cerebral grey and white matters. The dominating effects are: ma ssiv e va cuolisat ion of neurocytes and spongious ch anges in th e white matter.

2. Subacut e and chronic poisoning with mercury phenyla cetate brings about a considera ble increase of TPPase activity in the nerve cells of basal ganglia and of th e cerebral and cerebellar cort ex. Man y oligodendrocyt es of the white matter become rea ct ive with respect t o TPPase following intoxication with mercury ph enyla cetate. 3. Th e activity of cerebral acP and ATPase levels down following shor t term intoxicat ion with mercury phenylacetate to disappear entirely in many regions of the brain following prolonged administration of the poison. 4. Acetylcholinesterase activity is only slightly affe ct ed by mercury phenylacetate int oxication as indicated by th e moderately decreased staining for AChE in the neuropil of basal ganglia, the Amm on' s horn, the pons and oblongate medulla. 5. Th e degree of morphological and histoenzymic changes increases with the len gt h of time of the experiment. 6. Th e act ivit ies of cerebral NsE and alkP in either of the experiment al gro ups remain un affect ed by the intragastric application of mer cur y ph enyla cetate.

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