Cysteine Sulphinate and Cysteate: Mediators of Cysteine Toxicity in the Neonatal Rat Brain

European Journal of Neuroscience, Vol. 5, pp. 1398-1412 0 I993 European Neuroscience Association

Cysteine Sulphinate and Cysteate: Mediators of Cysteine Toxicity in the Neonatal Rat Brain?

Anders Lehmannl, Henrik Hagberg', Owe Orwar1v2 and Mats Sandbergl

Goteborg, Sweden Department of Anatomy and Cell Biology and 2Department of Analytical and Marine Chemistry, University of Goteborg,

Key words: excitotoxicity, MK-801, cerebrospinal fluid, glutamate, degeneration, amino acids, cerebral bleedings

Abstract

Excitotoxic amino acids contain two acidic groups, but cysteine represents an exception to this rule. The hypothesis that cysteine toxicity is mediated by the oxidized and diacidic metabolites cysteine sulphinate andlor cysteate was tested in the present study. The issue was approached in three different ways. Firstly, the distribution of brain injury after subcutaneous administration of cysteine (1 mg/g) to 4-day-old rats was compared with that caused by cysteine sulphinate (3 mglg). Secondly, the effects of excitatory amino acid receptor antagonists on cysteine and cysteine sulphinate toxicity were investigated. Thirdly, the cerebral concentrations of cysteine sulphinate were determined after cysteine administration and compared with those obtained after cysteine sulphinate injection. The cerebral cortex was the region most vulnerable to cysteine toxicity, followed by the hippocampus (especially the medial subicular neurons), amygdala, caudoputamen, cerebellum and septum. Pronounced extravasation of red blood cells was observed in lesioned areas. One day after cysteine administration, the injury was infarction-like and sharply demarcated. Cysteine sulphinate- induced damage resembled cysteine-induced lesions in some respects: the anterior cingulate and retrosplenial cortices, as well as medial subicular cells, were quite vulnerable. However, the differences prevailed. Cysteine sulphinate, but not cysteine, killed neurons of the superficial part of the tectum, the medial habenula, the ventromedial hypothalamus and the arcuate nucleus. Further, while cysteine toxicity was prominent in deep cortical layers, cysteine sulphinate preferentially damaged superficial cortical neurons. Cysteine toxicity was abolished by pretreatment with MK-801, a selective NMDA antagonist, but not by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, a selective AMPA receptor blocker. In contrast, the considerably smaller lesion seen after cysteine sulphinate administration was only partially prevented by MK-801. Large (19-fold) increases in cortical cysteine sulphinate concentration were noted after injection of a toxic dose of cysteine. This corresponds to 90 nmol cysteine sulphinatelg protein. The cysteate concentration was not increased above the detection limit. Injection of a toxic dose of cysteine sulphinate elevated cysteine sulphinate concentration in the frontomedial cortex (a region consistently injured by cysteine sulphinate) almost three orders of magnitude more than that observed after cysteine administration. Taken together, these results strongly suggest that neither cysteine sulphinate nor cysteate alone mediate cysteine toxicity. Possible candidates for such a role include cysteine carbamate and S-sulpho-cysteine. Another alternative is that the sulphydryl group of cysteine sensitizes the NMDA receptor complex and thereby enhances the effect of glutamate andlor some other endogenous NMDAmimetics.

Introduction Most studies pertaining to L-cysteine in the brain have focused on its role as a constituent of glutathione. Recent observations suggest that this amino acid may also serve neurotransmitter functions since it is released from brain slices in a Ca2+-dependent manner upon depolarization (Keller et al . , 1989; Zangerle ef al . , 1992) and since it excites neurons (Olney et al., 1990). This notion is of particular interest because cysteine has been identified as an excitotoxin. Despite the fact that this finding was made more than two decades ago (Olney

and Ho, 1970), surprisingly little is known about its toxic properties. In initial studies, lesions to hypothalamic and retinal neurons after peroral or parented administration of cysteine were demonstrated (Olney and Ho, 1970; Olney et al., 1971). It was subsequently shown that 24 h following a subcutaneous injection of cysteine in neonatal rodents, widespread damage chiefly affecting the forebrain develops (Olney et al., 1972; Sharpe et a l . , 1975). In support, newborn rats injected with cysteine exhibit advanced cerebral atrophy,

Correspondence to: Anders Lehmann, Preclinical Research and Development, Gastrointestinal Pharmacology, Astra Hassle AB, S-43 1 83 Molndal, Sweden

Received 23 March 1993, revised 18 May 1993, accepted 3 June I993

Cysteine neurotoxicity in the immature brain 1399

especially of the cerebral cortex, 1 month after injection (Lund Karlsen et al . , 1981).

Reports on the relative vulnerability of different brain regions to cysteine are to some extent in disagreement. Histopathological evaluation of brains fixed 24 h after cysteine administration suggests the following order of vulnerability: hippocampus > amygdala > cortex>thalamus (Sharpe et al . , 1975). However, long-term survival experiments indicate that the cerebral cortex is considerably more sensitive than the amygdala and the hippocampus (Lund Karlsen et al . , 1981). The discrepancies may partly be explained in terms of the dynamics of the development of the brain lesions, i.e. 24 h after injection the lesion may not be fully expressed, but at 30 days secondary events such as trans-synaptic degeneration may complicate the interpretation. In view of the discrepant reports, the distribution of cysteine-induced damage was reinvestigated in the present study. In the immature rat brain, the toxicity of cysteine can be abolished

by (+)-5-methyl- 10,11dihydro-5Hdibenzo(a,d)cyclohepten-5,lO-imine maleate (MK-801), a specific blocker of the ion channel linked to NMDA receptors (Olney et al., 1990). At low concentrations, cysteine acts preferentially on NMDA receptors in embryonic chick retinal neurons in vitro, while both NMDA and non-NMDA receptors are activated at higher concentrations (Olney et al . , 1990). In the present work, the effects on cysteine toxicity of the blood-brain barrier-penetrating non-NMDA antagonist 2,3-dihydroxy-6-nitro- 7-sulphamoyl-benzo(F)quinoxaline (NBQX) were evaluated and compared with those of MK-801.

The mechanism of action of cysteine is uncertain as the amino acid does not seem to fulfil the structural requirement for an excitotoxin, i.e. it lacks a second acidic group in addition to the a-carboxylate. Whereas it is possible that the carbamate of cysteine that is thought to form (Nunn et al., 1991) in the presence of bicarbonate/COz is toxic (Olney et al., 1990), an alternative hypothesis (Olney et al., 1971) suggests that cysteine is oxidized to cysteine sulphinate (CSA), which is both excitatory and neurotoxic. Another candidate as a mediator of cysteine neurotoxicity is the oxidized CSA congener cysteate (Olney et al., 1971). However, it is unknown if the immature brain can convert cysteine to CSA and cysteate, and the activity of the oxidizing enzyme, cysteine dioxygenase, is low in the neonatal central nervous system (Misra and Olney, 1975). The present study examines the effects of cysteine administration on brain concentrations of CSA and cysteate in neonatal rats. In addition, the total tissue concentrations of the cysteine metabolites alanine and taurine, as well as other amino acids, were measured. The levels of sulphur- and non-sulphur-containing amino acids were determined in brains from rats injected with a protective dose of MK-801 prior to cysteine administration. This experiment would provide information on whether changes in amino acids are related to NMDA receptor stimulation or to non-receptor mediated effects of cysteine.

In the embryonic chick retina, cysteine mimics NMDA with regard to the topographical and pharmacological profiles of the toxicity, but CSA is glutamatomimetic (Pullan et al., 1987). This is not evidence against CSA and cysteate playing a mediator role in cysteine toxicity in the mammalian brain: in the immature rat hippocampal slice (Lehmann and Jacobson, 1990), as well as in the neonatal murine arcuate nucleus (Lehmann and Jonsson, 1992), glutamate neurotoxicity is exclusively mediated by NMDA receptors. Further, glutamate damage to mature arcuate neurons can largely but not completely be prevented by NMDA antagonists (Lehmann and Jonsson, 1992). Thus, the isolated embryonic chick retina is not representative of the mammalian brain in this respect. Further, the theory that cysteine carbamate mediates cysteine toxicity is based on the bicarbonate

dependence of cysteine toxicity (Olney et al., 1990). However, bicarbonate plays a critical role in the toxicity of other excitotoxins in the embryonic chick retina (Zeevalk et al., 1989), so the carbamate hypothesis does not rule out a role for CSA and cysteate.

If cysteine toxicity is mediated by CSA and/or cysteate, the relative vulnerability of various neuronal populations should be similar after administration of the toxins. More importantly, the toxicity of both agonists should show the same sensitivity to receptor antagonists. The present study was consequently designed to compare the in vivo toxicity of cysteine and CSA.

Materials and methods In all experiments, 4-day-old (day of birth = day 0) Sprague - Dawley rats of either sex were used. The pups were lightly anaesthetized with ether to facilitate the injection and to reduce discomfort caused by the injection. Cysteine (50 mg/ml, dissolved in distilled water and titrated with NaOH to pH 6-7) was given at 0.5, 0.8 or 1.0 mglg S.C. MK-801 (1 pg/g) or NBQX (15 or 30 pg/g) was administered 20 -30 min before cysteine. Isotonic saline was given to cysteine controls. The control animals used for biochemical measurements were injected with an equal volume of saline or with MK-801 (1.0 pg/g). All solutions were prepared immediately before the experiments, a measure of particular importance for cysteine, which is spontaneously oxidized to cystine. The pups were allowed to recover for -30 min before they were returned to the dam.

A neutralized CSA solution was injected at 2.0 or 3.0 mg/g S.C. In some cases, MK-801 was administered at 1 .O pg/g 20-30 min prior to CSA injection.

Histopa thology In order to determine the early development of the injury, 4-day-old rats were fixed 6 or 24 h after cysteine administration. The 6 h survival period was chosen for two reasons. Firstly, biochemical measurements (see below) were made at this time and it was therefore relevant to describe the corresponding pathological changes. Secondly, certain subregions were completely destroyed after 24 h, but with a shorter survival time it was possible to identify gradients of vulnerability within these subfields. Survival for 24 h made it possible to study the injury when moribund neurons were still present.

The animals were deeply anaesthetized with diethylether and perfusion-fixed through the aorta with 12 ml of 2.5 % glutaraldehyde/ 2 .O% paraformaldehyde in phosphate-buffered saline. The brains of five pups were immersion-fixed after 24 h. In addition, the brains of two animals were fixed after 48 h. Pups injected with CSA were fixed after 17 rather than 24 h since CSA-induced damage developed quicker than cysteine-evoked injury. One group of animals were fixed 6 h after injection of CSA (3 mg/g). The brains of perfusion-fixed rats were removed from the skull and immersion-fixed overnight. After dehydration in graded ethanols, the tissue was embedded in glycolmethacrylate or paraffin. Plastic-embedded material was sectioned at 1 pm and stained with toluidine blue or Richardson’s stain, and paraffin-embedded tissue was cut at 3 pm and stained with haematoxylin/eosin.

Specific gravity Measurement of specific gravity was performed to quantify the oedema induced by cysteine. The following groups of animals were included: saline-injected controls, cysteine (1 .O mglg), cysteine plus MK-801 (I pglg) and cysteine plus NBQX (15 pglg). Twenty-four hours after

1400 Cysteine neurotoxicity in the immature brain

the injections, the animals were decapitated and their brains were placed in ice-cold kerosene. Biopsies from the cingulate - parietal cortex of frontal sections and from the temporal half of the hippocampus were isolated in ice-cold kerosene. The specific gravity of the samples was determined in a bromobenzene - kerosene specific gravity gradient which calibrated with 10 pl drops of K2S04 solutions of known specific gravity (Nelson et al., 1971). Measurements were done with duplicate samples, and readings were made 5 min after immersion of the biopsies in the gradient. The gradient was cleared after immersion of biopsies from three brains, and it was then recalibrated.

Amino acids: sample preparation Pups were injected with saline or 1.0 mg/g cysteine and killed by decapitation 1, 3 or 6 h later. For each of these groups, littermates served as controls. The changes in amino acids due to metabolism of cysteine, or to effects of cysteine unrelated to stimulation of the NMDA receptor, were assessed in animals injected with MK-801 (1.0 pglg) 20-30 min before cysteine administration (time of killing, 6 h). Littermates administered MK-801 served as control. The brain was rapidly isolated, and a biopsy corresponding to -0.5 mg protein was taken from the frontal cortex. This region was chosen since it was consistently damaged, and because it was easy to isolate rapidly, thereby limiting postmortem artefacts. The tissue was immediately immersed in N2(1). To prevent oxidation of cysteine, the biopsy was sonicated in ice-cold methanol (90%) containing 1 mM EDTA and 20 mM 2-mercaptoethanol. The extract was centrifuged at 16 OOO g for 20 min. The supernatant was stored at -80°C and the pellet was dissolved in 1 M NaOH for protein determination using bovine serum albumin as standard (Lowry et al., 1951). Amino acid concentrations were expressed relative to protein rather than to wet weight as there was pronounced cerebral oedema in cysteine-treated rats.

CSA was administered at 3.0 mg/g and the animals were decapitated 1 or 3 h later. The frontomedial cortex (a vulnerable region) was isolated bilaterally; in addition, the right ventral cortex (a resistant area) was isolated. The biopsies were processed as described above.

Amino acids: analysis Amino acid concentrations were determined using automated reversed phase HPLC separation of o-phthalaldehyde (OPA)-2-mercaptoethanol derivatives followed by fluorescence detection (Lindroth et al., 1985; Sandberg et al., 1986). Cysteine was alkylated with iodoacetate to form S-carboxymethylcysteine before derivatization with OPA. In contrast to cysteine, the resulting isoindole has a fluorescent intensity comparable with other amino acid -0PA adducts. The S-carboxymethylcysteine derivative eluted between aspartate and asparagine. Since 2-mercaptoethanol was used in the extraction procedure, any cystine present would be reduced to and measured as cysteine.

Determination of the concentrations of CSA and cysteate in the cortex of rats injected with cysteine was performed essentially as described earlier (Orwar et al., 1991) with the following modifications. The reversed phase columns used were either a 1 5 0 ~ 4 . 6 mm (lengthx internal diameter) or a 2 5 0 ~ 4 . 6 mm column (TSK ODS-80 TM, Tosoh, Tokyo) equipped with precolumns. Separation of acidic sulphur amino acids and y-glutamylpeptide derivatives was achieved with gradient elution using sodium citrate buffers (pH 4.67 or 4.83) and methanol (80%), acetonitrile (18.5%) and tetrahydrofuran (1.5%, HPLC grade, Rathburn Chemicals, UK) as mobile phases. Fluorescence detection was performed using an excitation wavelength of 230 nm, which increased signal/noise ratio - 5-fold compared to excitation at 330 nm. All fluorometric data were acquired and processed using Maxima 820 software (Waters, Dynamic Solutions, MA, USA).

Measurement of CSA in the brains of CSA-injected rats was done with the standard technique (Lindroth et al., 1985; Sandberg et al., 1986), as interest was focused on levels of CSA obtained after CSA administration rather than basal concentrations (which cannot be quantitated adequately using the standard method).

Chemicals MK-801 was a gift from Dr K. Rudolphi, Hoechst, Germany, and NBQX was a gift from Dr T. Honork, Novo Nordisk, Denmark. y-Glutamyl-Cys was from Dr E. Pileblad, Department of Pharmamlogy, University of Goteborg, Sweden, and cysteine and CSA were purchased from Sigma, St Louis, MO.

Results

Mortality All animals used for the biochemical and short-term pathology experiments survived the experimental period, except for one rat given MK-801 and one rat administered NBQX. The mortality rates reported below concern the animals used for histopathological evaluation and specific gravity determination 24 h after injection.

None of the 16 rats injected with cysteine at 0.5 mglg died, while two of the 17 animals given 0.8 mg/g died overnight (7-20 h post injection). The mortality rates for rats injected with 1.0 mg/g cysteine, cysteine + MK-801 (1.0 pglg) and cysteine + NBQX (15.0 pg/g) were 32% (10/31), 17% (2/12) and 60% (9/15), respectively. Only one of five rats administered cysteine + 30 pg/g NBQX survived for 24 h. Although it is not known whether NBQX given alone would produce a similar mortality rate, the probable cause of death after cysteine + NBQX was the marked depressive effect of the antagonist on breathing frequency observed even before administration of cysteine.

Administration of CSA at 2.0 mg/g did not result in any deaths (n = 5). One of nine rats given 3 .O mg/g died overnight, while all seven animals injected with 1 pg/g MK-801 before 3.0 mg/g CSA survived.

Beha viour The behavioural effects of cysteine were not quantified with objective methods but were so consistent that they deserve mentioning. At 0.5 mg/g, cysteine only slightly stimulated gross motor behaviour. Injection of 0.8 or 1.0 mglg cysteine induced hyperactivity within 1 h. This effect progressed to episodes of ‘wild running’ which possibly reflected epileptifonn activity. Hyperactive behaviour subsided 3 -4 h after cysteine administration. Pups given MK-801 and NBQX were markedly lethargic at the time of cysteine administration. MK-80 1 inhibited the cysteine-induced hyperactivity, but did not consistently abolish it. In contrast, NBQX delayed the appearance of the abnormal behaviour caused by cysteine, but once it had commenced the hyperactivity did not seem different from that observed in cysteine-injected controls.

The effects of CSA on behaviour were less consistent, but in general, animals injected with CSA underwent periods of wriggling movements. In addition, postural control was disturbed by CSA.

His topa thology

Cysteine-induced lesions Six hours after injection of cysteine (1.0 mg/g), there was marked swelling of neuronal perikarya and neuropil. This was most evident in the dorsolateral and medial parts of the cerebral cortex (Fig. lA), whereas the ventral cortex was unaffected. The outer cortical layers


FIG. 1. Six hours after injection of cysteine, the nuclei of neurons of the medial frontal cortex are rounded with dark chromatin and the neuropil is oedematous (A). Slit-like vacuoles are present in some neurons (arrowheads). The cortical toxicity is blocked by MK-801 (B) but not by NBQX (C). In the lateral subiculum (D), a few cells show advanced degenerative changes (arrows). The morphology of the majority of the neurons is only moderately altered, although these cells are moribund (cf. Fig. 2C). A small number of granule cells of the infrapyramidal blade and crest of the dentate gyrus are pyknotic (arrowheads in E). Final magnification: A-C: 340X; D: 382X; E: 2 3 4 X .


FIG. 2. Twenty-four hours after cysteine administration, there are no surviving neurons in the deep dorsomedial cortex (A). The neuropil is undergoing dissolution, and macrophages (arrow in A) are common. Cells in the interface between the intact outer layers and the infarcted inner layers are extremely swollen (B) and extravasated erythrocytes are present (arrow in B). At this time. all subicular neurons (C) are lysed. The border between necrotic and intact tissue is sharp (arrows in D and E). Note the rarefication of the neuropil corresponding to the localization of dendrites of damaged pyramidal cells (D and E). A focal haemorrhage is seen in the otherwise intact medial CAI region (encircled area in E). Final magnification: A, C, 340x; B, 382x; D, 128x; E, 68x.

were much less affected than the deep layers, although a small number of swollen neurons could occasionally be found among normal cells. The molecular layer was rarefied in the cingulate-parietal cortex, and

more markedly so at the rhinal fissure. Swelling was also prominent in the subiculum and strata oriens and pyramidale (Fig. 1D) of medial CA1, but it disappeared laterally. Oedema was present in the stratum


oriens in the CA3 region. The molecular layer of the infrapyramidal blade of the dentate gyrus and of the crest was rarefied (Fig. 1E) but the suprapyramidal blade was unaffected. Other regions showing oedematous reactions were the dorsolateral thalamus, the amygdala and the dorsolateral caudoputamen. Perikaryal changes ranged from swelling and slight clumping of chromatin to advanced damage reflected by large vacuoles and shrunken nucleus with doughnut-shaped chromatin (Fig. ID). The majority of injured cortical neurons displayed abnormally dark and round nuclei without focal chromatin contraction (Fig. lA, C). Slit-like vacuoles were often found in cortical neurons (Fig. lA, C). The degenerative process was most evident in the deep layers of the cingulate cortex, the indusium griseum, the subiculum and the infrapyramidal dentate granule cells. The latter population was always affected, but the number of injured cells was quite small (Fig. 1E). Thalamic neurons showed mainly mild alterations at 6 h. Haemorrhages were found predominantly in the outer cortical layers and in the subarachnoidal space. The early neuropathological changes mirrored largely the frank degeneration developing within 24 h (see below). However, the 6 h period was insufficient for identification of all moribund neurons. The shorter survival time made it possible to identify a gradient of vulnerability among moribund cells in the subiculum and lateral CA1; medial (Fig. 1D) but not lateral neurons showed degenerative changes after 6 h. The former population was thus more vulnerable, a finding that would have been overlooked using a 24 h survival period only.

After 24 h, many neurons in the regions mentioned above were in a late stage of degeneration, as evaluated in nine animals. The ventral cortex was always intact. The region most severely affected was that around or immediately dorsal to the rhinal fissure. The lesion was often sharply demarcated and infarction-like. The outer layers were usually intact except for those in the vicinity of the rhinal fissure, which were completely degenerated. However, necrotic cells in other cortical areas were found interspersed among the undamaged neurons of the outer layers. One consistent observation was that there were numerous extremely swollen cells in the interface between necrotic and normal neurons (Fig. 2B). This type of injury appeared quite different from that seen in the deep layers: while the nuclei of degenerating cells of the inner layers (Fig. 2A) were severely pyknotic with dense clumping of the chromatin, or karyorrhectic, the nuclei of dying cells of the outer cortex did not contain clumped chromatin but were so swollen that they were only faintly stained.

The hippocampus and the subiculum (Fig. 2C) were always damaged 24 h following cysteine administration. The lesion consistently extended in a mediolateral direction, thereby affecting the medial (Fig. 2D, E) but never the lateral CAI. Another vulnerable region of the hippocampus was the infrapyramidal blade of the dentate gyrus. Injured granule cells were scattered in the stratum granulare, but the suprapyramidal cells were never damaged (Fig. 2D, E). The injury to granule cells was most obvious at the septal and temporal poles, but total obliteration of granule cells was only seen in the latter. CA3 pyramidal cells were usually intact in the septal parts of the hippocampus, but they were always injured at more temporal levels (Fig. 2E). The CA4 region was normal (Fig. 2E) except in severe cases.

The third most vulnerable areas were the caudoputamen, thalamus and amygdala. In the caudoputamen, the lesion was usually confined to the dorsolateral regions, but it was sometimes diffuse. The lesion in the thalamus involved superficial dorsolateral nuclei, but extended occasionally to deeper nuclei. Among vulnerable regions, the septum, olfactory bulb and cerebellum were least susceptible. Damage was never found in the arcuate nucleus, habenular nuclei or tectum.

Cysteine produced cerebral haemorrhages that were readily observed

after 6 h and were considerably more pronounced after 24 h. Haemorrhages were most prominent in the outer layers of the dorsolateral cerebral cortex (Fig. 2B), i.e. in the layers where neuropathological alterations were least conspicuous. The subarachnoidal space was filled with red blood cells, which were also often present in the ventricles. There was no obvious relationship between the distribution of neurotoxicity and blood cell extravasation within injured regions (Fig. 2E). In five of seven animals injected with saline and subsequently perfusion-fixed, small haemorrhages were present in the cortex. Immersion-fixed brains of five rats administered cysteine exhibited haemorrhages, the severity and distribution of which could not be distinguished from perfusion-fixed material.

Phagocytosing and other inflammatory cells were numerous in all lesioned areas 24 h after cysteine administration (Fig. 2A). In particular, foam cells were common. It should also be mentioned that 24 h after cysteine administration, the lesion involved axon bundles such as the corpus callosum, which appeared partially disintegrated.

In two animals which were immersion-fixed 48 h after cysteine injection, moribund cells had largely been cleared, and the lesion showed all the characteristics of an infarction (degeneration of vascular elements, cystic cavities). Large numbers of macrophages were present, many of which contained phagocytosed red blood cells. In addition, the haemorrhages were almost completely resorbed at this time.

In a further attempt to rank the order of vulnerability, 17 pups were injected with 0.8 mg/g cysteine. All of the 15 survivors had cerebral haemorrhages. In three animals, the lesion was essentially as severe as in the rats administered 1 .O mg/g of cysteine. There was selective injury in the cingulate cortex in one animal, while four rats had lesions confined to the cingulate -medial parietal and retrosplenial cortices and to the cortex around the rhinal fissure. The remaining seven rats showed a similar distribution of cortical injury; in addition there was a loss of medial subicular neurons.

Of 16 rats injected with 0.5 mg/g cysteine, only one showed cerebral pathology. In this animal, there was a selective lesion in the cingulate- parietal cortex and indusium griseum.

Cysteine sulphinate-induced lesions In order to determine whether there was a difference in the timecourse of development of brain damage after cysteine and CSA administration, pups given CSA (3.0 mg/g) were fixed 6 h after injection (n = 3). The distribution of damage was similar to that observed 17 h after administration (see below). For instance, in contrast to the observations made on rats administered cysteine, the extent of the subicular lesion did not depend on the postinjection survival time in CSA-treated rats (compare Figs 3F and 4A). However, the necrotic process was, not unexpectedly, more advanced after 17 h than after 6 h.

There was neither any mortality nor any brain damage (except for a lesion in the arcuate nucleus of the hypothalamus) in pups given 2.0 mg/g CSA (n = 5; survival time 17 h). The occurrence of brain lesions after CSA is summarized in Table 1. Four of eight surviving rats injected with 3.0 mglg CSA carried a lesion in the olfactory bulb. The subiculum in direct contact with the third ventricle was consistently damaged (Fig. 4A) but the lesion was considerably smaller than that developing after 1.0 mglg cysteine. Damage to the dentate gyms granule cells was severe in the crest and medial infrapyramidal blade at septal levels where all neurons were killed, i.e. CSA-induced injury was, in comparison with cysteine-induced damage, more severe to granule cells than to subicular neurons (pathology at 6 h is shown in Fig. 3E and F). Cortical lesions were found in the dorsomedial frontal cortex (pathology at 6 h is shown in Fig. 3A). At more rostral coordinates, damage was also present at ventromedial surfaces, and


at the anterior pole of the frontal cortex, the lesion encompassed the entire medial region. Intracortical haemorrhages and inflammatory cells were commonly seen. Posteriorly, the lesion disappeared but reappeared in the posterior cingulate cortex, in the retrosplenial area and in the immediate vicinity of the rhinal fissure. Small focal lesions were occasionally present in the superficial region of the parietal cortex. The intracortical distribution of injury resembled that induced by cysteine in that the most superficially situated neurons were less vulnerable than those more deeply located, except for in the region

around the rhinal fissure, where the latter population also degenerated. In sharp contrast to the intracortical distribution of cysteine-induced damage, the lesion inflicted by CSA never extended to the deepest layers.

The arcuate nucleus and ventromedial hypothalamic neurons (Fig. 4C) degenerated after CSA administration. Another difference compared with cysteine was that CSA killed neurons of the medial habenular nucleus (Fig. 3D) and the dorsal (Fig. 4D) and to a smaller extent the lateral superficial aspects of the tectum. Lesions of the internal granule cell layer of the cerebellum were more pronounced after CSA


FIG. 3. (A) Degeneration of the frontomedial cortex 6 h after injection of cysteine sulphinate. Lateral aspects are preserved, and the oedema is mostly well demarcated (arrow in A). A number of dark neurons (arrows in B) can be observed in the border zone between oedematous and intact cortical tissue. In the medial parts of the frontal cortex, the neuropil is extremely swollen (C). Neuronal nuclear changes range from mild chromatin clumping (long arrow in C ) to more severe chromatin condensation in the periphery (short arrow in C). Some neurons contain a pyknotic nucleus (unfilled arrow in C). Only medial habenular neurons (D) facing the third ventricle succumb after cysteine sulphinate injection (CP; choroid plexus). Already 6 h after cysteine sulphinate administration, the crest and medial infrapyramidal granule cells of the dentate gyrus are killed (E). The lesion is conspicuously demarcated (arrows in E). Superficial dorsomedial thalamic neurons are also damaged (arrowhead in E). At this time, medial subicular cells display advanced necrosis whereas more lateral neurons survive. Arrow in E shows the border betweendying and undamaged cells. cc, corpus callosum. Final magnification: A, 68x; 8, 340x; C, 382x; D, 170x; E, 85x; F, 234x.

than after cysteine. The dorsal thalamus was damaged in four of eight animals injected with CSA, and this lesion was more clearly confined to the superficial regions than that caused by cysteine.

Effects of receptor antagonists The brains of two rats administered MK-801 prior to cystehe appeared normal when fixed 6 h after cysteine administration (Fig. 1B).


Pathological changes in animals fixed 6 h after injection of 15 pg/g NBQX before cysteine were indistinguishable from those in cysteine-treated controls (compare Fig. 1A and D). For instance, in cysteine controls (n = 5), 29&6% of the neurons of the indusium griseum contained nuclei with conspicuous chromatin clumping. The corresponding figure for animals given NBQX + cysteine (n = 4) was 24 &2%, (P > 0.05, Student’s t-test). Three of four rats administered MK-801 prior to cysteine did not develop any cerebral pathology in 24 h, and one showed a discrete lesion confined to the cingulate and medial parietal cortices and to the indusium griseum. MK-801 also prevented cysteine-induced haemorrhage. The brains of three pups given NBQX (two rats received 15 pg/g and one was given 30 pglg) before cysteine (fixation time 24 h) could be distinguished from cysteine-injected controls neither in terms of severity or distribution of the injury, nor in terms of haemorrhages.

MK-801 abolished macroscopic haemorrhages caused by CSA (3.0 mg/g). A comparison of the damage in CSA-injected animals with their littermates given MK-801 prior to CSA clearly showed that the antagonist ameliorated, but generally failed to abolish, the toxicity of CSA (Table 1). The arcuate nucleus was better protected by MK-801 against CSA toxicity than lateral hypothalamic nuclei. In contrast to the complete protection afforded by MK-801 against cysteine toxicity in the subiculum, the antagonist never abolished the much more limited subicular injury inflicted by CSA (compare Fig. 4A and B).

Changes in specific gravity The specific gravity of the cerebral cortex and hippocampus was significantly reduced 24 h after cysteine administration (Table 2). The values obtained in animals injected with cysteine and MK-801 were not different from controls. NBQX did not significantly affect the cysteine-induced decrease in hippocampal specific gravity. However, the specific gravity of the cortex was lower in animals administered both NBQX and cysteine compared with animals given cysteine only.

Effects of cysteine on brain amino acids There was some variation between the amino acid concentrations in the different groups of saline-injected controls which could be due to litter differences and/or to variations in sample preparation and analysis. Consequently, amino acids were expressed as percentages of litter- matched controls, the cortical extracts of which were analysed on the same occasion. However, in order to provide an estimate of control levels, data pooled from all control groups are presented in Table 3. Cysteine administration reduced the levels of glutamine, valine, isoleucine and leucine after 1 h (Table 3). After 3 h, taurine was significantly reduced and glutamine was further decreased while most other amino acids were increased. Six hours after cysteine injection, the majority of amino acids were elevated. However, taurine and glutamine were 76% and 38% of control values, respectively. The concentration of some amino acids, for instance aspartate and serine, never deviated from the control concentration. Cysteine increased rapidly and reached peak levels - 19 times basal concentrations 1 h after administration. The increase in CSA was delayed and amounted to 1763% of control after 6 h. In extracts from control as well as cysteine-injected animals, the concentrations of cysteate and of the cysteine-related excitants homocysteate and homocysteine sulphinate were below the detection limit (<200 fmol; Fig. 5).

One consistent finding after cysteine injection was an increase in a peak eluting before the OPA-y-glutamyl-cysteinyl-glycine derivative (Fig. 5). The relative increase 6 h following cysteine administration was comparable with that of CSA, but the control concentration was about ten times higher. This compound was identilied as y-glutamyl-Cys.

TABLE 1 . Effects of MK-801 (1.0 pglg) on injury induced by cysteine sulphinate (CSA, 3.0 mglg)

Region

Tectum

Subiculum

Dentate gyrus

Arcuate nucleus

Cortex Temporal at the rhinal fissure

Frontomedial

Posterior cingulate - retrosplenial

Cerebellum

Habenula

Degree of damage

severe moderate discrete absent severe moderate discrete severe moderate discrete absent severe discrete absent

severe discrete absent severe moderate discrete absent severe moderate discrete absent severe absent severe absent

Treatment

- CSA

418 118

318 718 118

418

~

418 818

418 118 318 618 118 118

518 118

218 418 41 8 418 418

CSA + MK-801 1 I7 117 217 317

417 317

1 I7 217 411

617 1 I7

1 I7 617 217

517 217

217 317

I17

717

The ratios represent the fraction of animals showing the type of damage indicated. The rat pups were perfusion-fixed 17 h after subcutaneous injection of CSA.

In preliminary experiments, primary cultures of rat cortical neurons were not injured by 24 h of exposure to 100 pM y-glutamyl-Cys as measured by release of lactate dehydrogenase (Lehmann and Thorkn, unpublished).

In order to determine how the cysteine-induced changes in amino acids relate to NMDA receptor mediated excitatiodtoxicity , animals injected with cysteine and MK-801 were compared with animals given saline and MK-801. This approach was also employed to examine whether cysteine-induced haemorrhages, which were accompanied by leakage of the blood-brain barrier, would increase the uptake of cysteine and promote formation of cysteine metabolites such as CSA and alanine. Six hours after administration, aspartate was halved, and glutamine was decreased by 26% (Table 4). The decrease in taurine observed after cysteine injection was blocked by MK-801, but the increase in alanine was unaffected. The augmentation seen for most other amino acids and ethanolamine was abolished by MK-801. In relative terms, cysteine increased twice as much as after injection of cysteine only, but this appeared to be caused by variations in control concentrations. Further, MK-801 seemed to inhibit the elevation of CSA, but again this was a result of fluctuations in basal levels. In this context, it should be stressed that the control levels of Table 3 represent pooled values from all saline-injected controls. Since the changes after cysteine injection are related to one specific control group, the absolute levels cannot be calculated from Table 3. Because the experiments were not paired, it cannot be excluded that MK-801 might affect amino acid levels.

The concentration of CSA in a vulnerable region, the frontomedial cortex, amounted to 34 pmol/g protein 1 h after CSA administration


FIG. 4. Seventeen hours after cysteine sulphinate injection, the medial subiculum is destroyed (A). However, if MK-801 is given before cysteine sulphinate, some neurons are spared (B). At this time, both the arcuate nucleus (arc) and the ventromedial hypothalamus (vmh) are necrotic (C). In the superior colliculus (D), superficial (open arrows) but not deeply located neurons are killed (ps, pial surface). Final magnification: A, B, 2 0 0 ~ ; C, 8 0 ~ ; D, 400~.

and 83 pmol/g protein 3 h after injection (Table 5). The corresponding values for the resistant ventral cortex were 14 and 56 pmollg protein, respectively.

Discussion Distribution and pharmacology of cysteine- and cysteine sulphinate-induced lesions The discrete haemorrhages found in some saline-injected controls suggest that the haemorrhages in animals perfusion-fixed after cysteine administration were artefactually large because of too high a perfusion pressure. However, the haemorrhages observed in five animals the brains of which had been immersion-fixed 24 h after cysteine injection were as severe as in the perfusion-fixed brains. This observation is in keeping with the high incidence of spontaneous cortical haemorrhage in 4-day-old rats (Pavlik and Mares, 1992). Haemorrhages induced

by cysteine were much more frequent in regions where neurotoxicity was pronounced, but there was no clear-cut colocalization within any given region, particularly not in the cortex. This does not necessarily imply that lesion sites were not a major focus of haemorrhages; the oedema evoked by cysteine might induce pressure gradients, forcing b l d cells towards the cortical surface. Cysteine-induced haemorrhages were probably secondary to neuronal degeneration since focal injection of NMDA into the immature rat brain also produces haemorrhages (Ikonomidou et al., 1989a).

In accordance with previous work (Olney er al., 1972), the dose - response relationship for toxicity of cysteine was steep. This observation is in agreement with earlier findings and seems to relate to the dose-dependency of cerebral uptake of cysteine (Misra, 1989). The results from the experiments with animals given MK-801 before cysteine eliminate breakdown of the cerebral bamers as an explanation for this threshold effect.


TABLE 2. Effects of cysteine and of cysteine in combination with MK-801 or NBQX on brain specific gravity

Specific gravity Hippocampus Cortex n

Control 1.0345 *0.0003 1.0346 f O.OOO4 5 CYS 1.0299 *0.0006'vb 1.0308*0.0004'~b~C 12 CYS + MK-801 1.0343+0.0002 1.0343*0.0007 4 Cys + NBQX 1.0274&0.0012 1.0289 kO.OOO6 5

Specific gravity was measured 24 h after injection of saline, cysteine (Cys, 1 mglg), Cys + MIL801 (1 pglg) or Cys + NBQX (15 pglg). The temporal half of the hippocampus and the frontoparietal cortex were used. "P < 0.01 for Cys versus control; bP < 0.01 for Cys versus Cys + MK-801; 'P c 0.05 for Cys versus Cys + NBQX (factorial ANOVA followed by Dunnett's r-test for multiple comparisons).

TABLE 3. Alterations in the concentrations of amino acids and amines in the frontal cortex of 4-day-old rats injected with cysteine (1.0 rnglg)

Amino acid Control Hours after cysteine administration (mine) &rnol/g protein) 3 6

CYS 0.56*0.06 1913*25oC 1228+ 18oC 1341*8oC CSA 8.1 +0.88 464+61' 1213*154' 1763t188' ASP 27.0 *2.1 110*18 111*26 83*7 Glu 73.9 * 5 . 5 103*7 140*11a 144*11b Ser 17.1 *1.3 100+17 129*12 145+27 Gln 47.6 ~ 2 . 5 85 k 3' 40*6' 38*2' Pea 81.3 *4.5 95*3 94*5 94 *4 Tau 320.4 +15.4 92+3 83+3b 76a5b Ala 13.6 *1.2 146 * 39 267 f 30b 283 + 3oC TYr 3.0 *0.3 73*24 328k29' 2 4 8 ~ 3 1 ~ GABA 13.4 *0.7 98*3 119+4b 115&6 Ea 1 . 1 *O.l 79+12 5 1 8 a W 595a45' Met 1.1 +0.1 n.d. 414*34' 460*41' Val 2.9 *0.2 55*9b 328a13' 322a33' Phe 3.4 *0.2 89+11 142*15' 119a6' Ile 2.1 *O.l 49+9b 263*28' 192+19b Leu 6.6 +1.3 58*14' 310+36' 103k13

The control values are. means t SEM pooled from three litters, n = 15. Changes reported are percentage changes relative to litter-matched controls, analysed on the same occasion. Therefore, the absolute values for each time point cannot be calculated from the pooled levels and relative changes. The number of controls and cysteine-injected animals were 5 and 5 (1 h) and 5 and 6 (3 and 6 h), respectively. hnollg protein; Ea, ethanolamine; Pea, phosphoethanolamine; CSA, cysteine sulphinic acid; n.d., not determined. .P <0.05; bP < 0.01; CP < 0.001.

The regional variation in vulnerability to cysteine differed somewhat from that reported by Sharpe et al. (1975), who found that the cortex was less susceptible to cysteine toxicity than both the hippocampus and the amygdala. The present findings show that the cortex was more sensitive than the hippocampus and the amygdala. The present results therefore agree best with the long-term studies of Lund-Karlsen et al. (1981).

Inasmuch as cysteine-induced brain damage was exclusively mediated by NMDA receptors, there should be some correspondence between the distribution of NMDA receptors and cysteine-induced injury. However, this was not the case. In the cortex, the density of NMDA binding sites is higher in the outer than in the inner layers (McDonald et al . , 1990a), yet the lesion was more pronounced in the latter. In support of earlier work (Sharpe et al., 1975; Lund-Karlsen et al., 1981), the subiculum was always more vulnerable than the CAI region, in spite of the finding that 4 days after birth there are approximately three times more NMDA receptors in the CAI (McDonald et al., 1990a). Even more remarkable was the observation that a small fraction of the infrapyramidal granule cells was damaged, whereas the

1

I 101 n1nut.s

FIG. 5. Chromatograms showing the elution profile for the standard ( 1 ) and cortical extracts from a control rat (2) and from a rat administered cysteine (3) 6 h before being killed. CA, cysteic acid; CSA, cysteine sulphinic acid; HCA, homocysteic acid; HCSA, homocysteine sulphinic acid.

suprapyramidal granules were always intact. In the 7-day-old rat hippocampal slice (Ellrkn and Lehmann, 1989), as well as in vivo (McDonald et aZ., 1990b; Young et al., 1991), granule cell vulnerability to NMDA is the opposite. That the suprapyramidal cells are more vulnerable is consistent with the finding that they are formed before their infrapyramidal counterparts, and therefore express more NMDA receptors in early development (McDonald et al., 1990a). Although vulnerability depends on many factors in addition to receptor density, differential vulnerability of hippocampal neurons to cysteine correlates best with neuronal proximity to cerebrospinal fluid. One implication of this is that cysteine may cross the blood-cerebrospinal fluid barrier better than the blood - brain barrier.

The relationship between vulnerability and cerebrospinal fluid does not apply to the cortex with one possible exception. In the cortex surrounding the rhinal fissure, the most superficial neurons were consistently killed after 24 h. It may be speculated that this was due to the larger volume of cerebrospinal fluid present in the fissure compared with the subarachnoidal space outside the rest of the cortex. In support of this notion, this cortical area is completely destroyed by an intraventricular injection of NMDA in infant rats (Lipartiti et al . , 1991). A delayed effect of cysteine was extreme swelling of outer cortical cells. Their unusual pathological features suggest that they


TABLE 4. Effects of cysteine on amino acid concentrations in the presence of MK-801

Amino acid MK-801 Cysteine + MK-801 X change n = 6 n = 7

CYS CSA ASP Glu Ser Gln Pea Tau Ala TYr GABA Ea Met Val Phe Ile Leu

pmollg protein 0.35 k0.05 10.24 &2.06b 8.3 k0.8a 71.9 k10.7b

27.7 +1.7 14.2 +0.5b 53.0 k3 .2 48.3 k1.2 14.6 *2.0 13.2 k l . 0 48.2 k3 .6 35.8 k2.2a 86.6 *3.1 78.1 k2 .9

315.2 k10.3 289.4 +8.9 7.9 k1.4 22.7 +2.0b 2.8 k0.2 3.2 k0 .2

10.6 +0.3 10.8 +0.4 0.68+0.03 0.67 *0.08 1 . 1 & O . l 2.0 kO.lb 4.9 *0.5 5.0 k0 .4 4.3 *0.2 3.7 *O.la 2.1 k0 .2 2.6 k0 .3 4.9 k0 .6 6.9 k0.5a

2825 766 - 49 -9 - 10 - 26 - 10

-8 187 14 2

-2 82 2

- 14 24 41

MK-801 (1 pglg) was administered S.C. 20-30 min before cysteine (1.0 mg/g, s.c.). The animals (both groups were from the same litter) were killed 6 h after cysteine injection, and extracb for amino acid analysis were prepared from the frontal cortex. The absolute amino acid levels of the MK-801-treated animals are not directly comparable with those of saline-injected controls (Table 3) because of litter differences and since extraction and amino acid analysis were done on separate occasions. The results are mean I SEM. aP < 0.05; bP < 0.001 (Student’s two-tailed, unpaired t-test); hmol/g protein.

TABLE 5. Levels of cysteine sulphinate (CSA, fimol/g protein) in the frontomedial and ventral cortices after injection of a dose of CSA (3 mglg) that causes cytopathology in the former but not latter region

Cortical region Time after CSA injection l h 3 h

Frontomedial Ventral

33.6+4.7 82.6+ 10.8 13.6k2.3 55.8k15.6

The results represent mean & SEM; n = 6 in both groups of pups, which were from the same litter.

succumbed secondarily to events other than direct NMDA receptor activation by cysteine. Massive haemorrhages producing local hypoxia might be one causative factor, and release of cytotoxic agents from underlying degenerating cells might be another.

Most interestingly, cysteine-induced brain damage in neonatal rats was reminiscent of that developing during severe hypoglycaemia in adult rats (Auer et al., 1984). Although disparities exist for some regions (e.g. the intracortical distribution), the relative vulnerability of hippocampal neurons is strikingly similar in the two conditions. Hypoglycaemic injury has been proposed to be caused by a cerebrospinal fluid-borne neurotoxin (for references see Auer et al., 1984). In the light of the fmding that neither glutamic acid nor aspartic acid increase the cerebrospinal fluid of hypoglycaemic rats (Simon et al., 1986), cysteine may be a candidate for a mediator role in hypoglycaemic brain damage, and CSA may be another (see below).

The relationship between neuronal vulnerability and proximity to cerebrospinal fluid was more clear cut for CSA than for cysteine. In particular, the complete destruction caused by CSA of the medial subiculum and of the crest and medial infrapyramidal blade of the dentate is identical to hypoglycaemic injury. The distribution of CSA-induced injury was indistinguishable from that produced by orally

administered glutamate in infant mice, a lesion which has been explained in terms of preferential uptake of glutamate at the choroid plexus (Lemkey-Johnston and Reynolds, 1974). In this context, it is of interest to note that hypobaric-ischaemic damage in the immature rat brain (Ikonomidou et al., 1989a,b) is very similar in most aspects to that inflicted by CSA (and glutamate). CSA produced brain injury that resembled cysteine-induced damage in some respects. However, certain areas were lesioned by CSA and not by cysteine, and the reverse was also true. In addition, the vulnerability of the cortex to the toxins differed. Due to this higher lipophilicity, cysteine probably penetrates the blood-brain barrier better than CSA. Further, there might be regional disparities in the uptake of the toxins over the blood-brain barrier. Consequently, the differential distribution of brain injury is not in itself a compelling proof against a mediator role for CSA in cysteine toxicity. The finding that the medial habenular nucleus, the hypothalamus and the tectum were damaged by CSA, but not by cysteine at a dose producing considerably more severe brain damage is puzzling. For instance, the medial habenular neurons are directly exposed to the cerebrospinal fluid of the third ventricle, they are killed by peroral glutamic acid (Lemkey-Johnston and Reynolds, 1974), and they degenerate in an NMDA-receptor mediated fashion afkr hypobaric ischaemia (Olney et al., 1989).

The present results corroborate a recent demonstration that cysteine is a selective NMDA agonist in vivo (Olney et al., 1990). In only one animal pretreated with MK-801 was a small cortical lesion present, while all animals pretreated with the blood - brain barrier-penetrating selective non-NMDA antagonist NBQX developed brain damage at least as severe as controls. The morphological findings were confirmed by specific gravity determinations which actually suggested that the cortical lesion was aggravated by NBQX. The specificity of cysteine as an NMDAmimetic might partly be due to the reducing properties of its sulphydryl moiety. Reducing agents have been shown to increase NMDA receptor mediated neurotoxicity in vitro (Aizenman er al., 1990), presumably by acting at a redox site in the receptor-channel complex. However, the specificity of cysteine as an NMDA agonist is not unique since glutamate shares this property (Lehmann and Jonsson, 1992).

MK-801 inhibited, but generally failed to completely block, the toxicity of CSA. Only regions with a high threshold for CSA toxicity were fully protected by MK-801. Interestingly, MK-801 was more effective in preventing toxicity in arcuate nucleus neurons than in nerve cells lateral to the arcuate. Cysteine produced considerably more severe damage in the subiculum than did CSA; nevertheless, only cysteine toxicity could be abolished by MK-801. It seems likely that CSA is a quite weak NMDA agonist with additional effects on non-NMDA receptors, a proposal in accordance with voltage patch-clamp studies (Patneau and Mayer, 1990). CSA toxicity would presumably then only be obliterated by co-administration of NMDA and non-NMDA antagonists. At high concentrations, cysteine produces an NMDA- and non-NMDA-receptor mediated lesion in chick embryonic neurons in vitro (Olney et al., 1990). In view of the present findings, it is possible that this type of damage in part is mediated by CSA.

Effects of cysteine on brain amino acids An early effect of cysteine was a decrease in some amino acids transported by the L carrier. It is therefore possible that cysteine, at high blood concentrations, inhibits the cerebral uptake of the large neutral amino acids. This effect was reversed after 3-6 h, i.e. at a time when the blood cysteine concentration was presumably declining. The elevation of many amino acids might be secondary to decreased protein synthesis and/or increased proteolysis, two effects known to


occur after excitotoxic stimulation (Siman and Noszek, 1988; Vornov and Coyle, 1991). In support, MK-801 blocked the cysteine-induced increase in a number of amino acids. Qualitatively, the effects of cysteine on brain amino acids are generally in agreement with a previous report (Misra, 1982). Some differences exist, possibly because the earlier report concerned amino acid concentrations in whole brain homogenates which included regions resistant to cysteine toxicity.

The massive NMDA receptor-mediated augmentation in ethanolamine might mirror hydrolysis of membrane phosphatidylethanolamhe in degenerating cells. Cerebral oedema mused by acute blood hyposmolality does not induce neural cell death, but has an identical effect on ethanolamine without affecting phosphatidylethanolamine levels (Lehmann et al., 1991). Therefore, the cysteine-evoked increase in ethanolamine might reflect metabolic alterations as well as membrane degradation.

Effects of cysteine on cysteine-related metabolites Cysteine is metabolized through two pathways in the central nervous system. Firstly, cysteine may be transaminated to yield 3-mercaptopyruvate and glutamate (at the expense of a-ketoglutarate). Secondly, cysteine may be oxidized to CSA, which is transaminated by aspartate aminotransferase to 3-sulphinyl pyruvate. This molecule decomposes spontaneously to pyruvate and sulphite, which is further oxidized to sulphate. Both reactions thus lead to formation of pyruvate and glutamate. The former can be transaminated to yield alanine and a-ketoglutarate, thereby consuming the glutamate formed. CSA may also be decarboxylated to hypotaurine or oxidized to cysteate, both of which are converted to taurine by oxidation and decarboxylation, respectively (see Huxtable, 1989 for review).

The effects of cysteine unrelated to neurotoxicity show that the increase in alanine, the mechanism of which is explained above, is matched by the decrease in aspartate. The aspartate aminotransferase reaction is normally directed towards aspartate synthesis (Fitzpatrick et al., 1983). Since CSA is a substrate for aspartate aminotransferase (Recasens and Benezra, 1981), the reaction may be reversed in cysteine-loaded animals. This proposed mechanism implies that high intracellular CSA concentration may be reached as the K, for the reaction is in the mM range. Indeed, the marked increase in alanine concentration is suggestive of a high metabolism of CSA to pyruvate by means of the transaminase reaction. The decrease in glutamine could be due to the powerful stimulation of glutaminase by sulphate (Bradford and Ward, 1976). The reason why glutamate was not increased is unknown, but it was proposed that it was consumed by glutamate dehydrogenase or released into the circulation. Cysteine is synthesized from methionine in the trans-sulphuration pathway. The increase in methionine after cysteine administration might reflect a smaller demand on methionine utilization.

Taurine is one end-product of cysteine catabolism and might therefore be expected to increase after injection of cysteine. Instead, the levels of taurine were suppressed, an effect caused by NMDA receptor stimulation. Since NMDA receptor activation is known to release taurine in vivo (Lehmann et al., 1984), it is likely that taurine was lost from the extracellular space into the circulatory system. The observation that taurine did not increase after co-administration of cysteine and MK-801 is consistent with the slow turnover of taurine in the brain (Huxtable, 1989).

Cysteine reached maximal concentrations 1 h after administration and declined thereafter. It has previously been reported that cysteine levels of whole brain peak 5 h after S.C. injections (Misra, 1989). MK-801 failed to inhibit the cerebral uptake of cysteine, which demonstrates that the breakdown of the blood - brain barrier did not further increase cysteine uptake.

The concentration of CSA in the neonatal rat brain found in the present study, as well as the failure to demonstrate any endogenous cysteate, agrees with a recent report in which precautions to prevent spontaneous oxidation of cysteine also were taken (Waller et al., 1991). Although the time elapsed between decapitation and extraction was < 1 min, it cannot be excluded that CSA concentration was underestimated due to transamination of CSA by aspartate aminotransferase (see above), which is very active in the neonatal brain (Benuck et al., 1971). The main biochemical observations were that CSA increased pronouncedly following cysteine administration, whereas the other cysteine-related excitotoxins cysteate, homocysteate and homocysteine sulphinate did not increase above the detection limit. As could be expected, the elevation of both alanine and CSA was delayed as compared with cysteine. The absolute levels of CSA were not significantly affected by MK-801 which excludes the possibility that the antagonist is neuroprotective by inhibiting formation of CSA.

The present results provide evidence against a mediator role of CSA (and/or cysteate) in cysteine excitotoxicity. The cerebral uptake of CSA must be assumed to be much lower than that of cysteine because the sulphinate group is more hydrophilic than the sulphydryl group. Therefore, most of the increase in cortical CSA after cysteine injection probably resulted from intracerebral production. Cysteine dioxygenase is an intracellular enzyme, and there is an efficient re-uptake system for CSA (Grieve et al., 1991). Thus, CSA was probably formed intracellularly , and its intracelluladextracellular gradient was maintained at a high level. Tissue CSA levels 6 h after cysteine administration were -90 nmol/g protein. If the protein concentration is approximated to 60 g/kg wet weight, the average tissue concentration would correspond to some 5 pM. Assuming further that the intracellular/extracellular gradient for CSA is similar to that for glutamate (i.e. at least 2000), one would arrive at mean extracellular levels of CSA in the nanomolar range. In the adult brain, extracellular glutamate is about three orders of magnitude higher, and as CSA and glutamate are equipotent toxins (Olney et al., 1971), it appears most unlikely that CSA reaches toxic levels in the extracellular space after cysteine administration. These calculations disregard the possibility that extracellular CSA may be present at much higher concentrations in certain subregions or in the synaptic cleft. Further, they do not take into consideration that CSA might be released in the critical phase of the injury, thereby accelerating the degeneration.

Administration of CSA was accompanied by a massive augmentation of cortical CSA concentration. The elevation of CSA was larger in the vulnerable frontomedial cortex compared with the non-vulnerable ventral cortex. This indicates that total tissue CSA can be present at levels 600-fold higher than those occuring after cysteine administration and still not produce any toxicity. The concentration of CSA in the sensitive frontomedial cortex was almost three orders of magnitude higher than that obtained after injection of cysteine. Again, these measurements do not discriminate between intra- and extracellular pools, but it is likely that the differences were even more pronounced in the extracellular space; after cysteine administration, most CSA was presumably generated within neural cells, while much of the CSA reaching the brain after CSA injection probably first passed the interstitial space.

If neither CSA nor cysteate acts as a mediator of cysteine toxicity, and if cysteine does not represent an exception with regard to the structure - activity relationship for excitotoxins, other candidates must be sought. One possibility is that cysteine forms a toxic carbamate in the presence of bicarbonate/COz (Olney et al., 1990). Alternatively, if the bicarbonate dependence of cysteine toxicity is unique to this amino acid (which remains to be shown), generation of S-carboxycysteine, which has a structure consistent with that of an excitotoxin (Max, 1991),

Cysteine neurotoxicity in the immature brain 141 1

may explain the effects of cysteine. However, the delay in the expression of cysteine toxicity (compared to CSA toxicity) suggests that a metabolic conversion rather than a non-enzymatic reaction accounted for the formation of the toxin. S-sulphocysteine, one of the most potent naturally occurring excitotoxins known (Olney et al., 1975), may be a strong candidate since toxic doses of cysteine possibly lead to sulphite accumulation and S-sulphocysteine synthesis. Further, we have recently found that S-sulphocysteine toxicity in the rat arcuate nucleus can be completely prevented by MK-801. Finally, the toxicity of cysteine may not be related to a metabolic product but rather to its sulphydryl moiety. Thus, it has been shown that the sulphydryl dithiothreitol kills cultured neurons by an NMDA receptor-mediated mechanism (Aizenman et al., 1990). Since this compound bears no structural resemblance to excitotoxins, dithiothreitol as well as cysteine may act by potentiating the effect of endogenous excitotoxins such as glutamate. Another possibility is that the sulphydryl group of cysteine chelates an anti-excitotoxic metal ion (Eimerl and Schramm, 1992) and thereby permits endogenous glutamate to overcome the threshold for excitotoxicity. In conclusion, CSA does not seem to act as a mediator of cysteine

toxicity since (i) the cerebral levels of CSA after injection of cysteine were probaby too low to be neurotoxic, (ii) the distribution of cysteine and CSA induced lesions differed in some important aspects, and (iii) cysteine toxicity could be fully blocked by MK-801, whereas the considerably smaller lesion caused by CSA could only be partially inhibited by the NMDA antagonist.

Acknowledgements This study was supported by the Swedish Medical Research Council (grant B92-12X-09053-03A to A. L.) and the Swedish Natural Sciences Research Council (grant 1905-307 to M. S.).

Abbreviations AMPA CSA EDTA GABA HPLC MK-801

NBQX NMDA OPA

c~-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid cysteine sulphinic acid ethylene diamine tetraacetic acid y-aminobutyric acid high-performance liquid chromatography (+)-5-methyl- 10,ll -dihydro-5H-dibenzo(a,d)cyclohepten- 5,lO-imine maleate 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline N-methyl-D-aspartate o-phthalaldehyde

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Cysteine Sulphinate and Cysteate: Mediators of Cysteine Toxicity in the Neonatal Rat Brain

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