Kynurenic acid synthesis by human glioma

Journal of the Neurological Sciences, 1990, 99:51-57 Elsevier

51

JNS 03389

Kynurenic acid synthesis by human glioma Annamafia Vezzani 1, Jan Bert P. Gramsbergen 2, Pietro Versari 3, Mafia Antonietta Stasi 1, Francesco Procaccio 3 and Robert Schwarcz 2 l lstituto di Ricerche Farmacologiche "'Mario Negri", Milano (Italy), 2Maryland Psychiatric Research Center, University of Maryland School of Medicine Baltimore, Maryland (U.S.A.) and 30spedale Niguarda, Department of Neurosurgery, Milano (Italy) (Received 18 January, 1990) (Revised, received 25 April, 1990) (Accepted 30 April, 1990)

SUMMARY

Biopsy material from human gliomas obtained during neurosurgery was used to investigate whether pathological human brain tissue is capable of producing kynurenic acid (KYNA), a natural brain metabolite which can act as an antagonist at excitatory amino acid receptors. Upon in vitro exposure to 40, 200 or 1000 #M L-kynurenine, the immediate bioprecursor of KYNA, freshly prepared tissue slices in a dose-dependent fashion produced KYNA which was detected in the incubation medium. De novo synthesized KYNA was identified by several chromatographic procedures. Astrocytomas produced significantly more KYNA than glioblastomas.

Key words: Excitotoxins; Glioma; Kynurenines; Kynurenic acid

INTRODUCTION

Kynurenic acid (KYNA) is a peripheral tryptophan metabolite, which has recently been identified as a constituent of the mammalian brain (Carlfi et al. 1988; Turski et al. 1988), and which can function as an antagonist at cerebral excitatory amino acid receptors (Perkins and Stone 1988). The realization that pharmacological blockade of these receptors affords neuroprotection in situations of experimentally induced neuroCorrespondence to: Robert Schwartz, Ph. D., Maryland Psychiatric Research Center, P.O. Box 21247, Baltimore, MD 21228, U.S.A. Phone: (301) 455-7635. 0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

52 degeneration (Schwarcz and Meldrum 1985) has led to the suggestion that a hypofunction of brain KYNA may be causally related to the nerve cell loss observed in neurological diseases (Schwarcz et al. 1984; Stone and Connick 1985). KYNA administration can indeed prevent or arrest neuronal death in animal models of cerebral ischemia and neonatal hypoxia (Andin6 et al. 1988; Simon 1988). Studies with rat brain tissue slices have recently provided evidence for the efficient production and subsequent effiux of KYNA upon administration of its immediate bioprecursor kynurenine (Turski etal. 1989). KYNA synthesis is catalyzed by kynurenine transaminase, an enzyme known to be present in rat and human brain (Minatogawa et al. 1974; Nakamura et al. 1988). Experiments with neuron-depleted brain tissue, which shows largely increased KYNA production at a time of pronounced astrogliosis, suggest that astroglia may function as the site of de novo synthesis of brain KYNA (Turski et al. 1989). We now report the results of a first attempt to assess the production of KYNA in human brain biopsy material, namely in glial tumors obtained during neurosurgery.

PATIENTS AND METHODS

Surgery and sample collection Removal of brain tumors was performed according to the standards of oncological brain surgery. Biopsies were carefully isolated and examined by a pathologist. The excised specimens were immediately placed in an ice-cold oxygenated Krebs-Ringer buffer solution (KRB), and the cold tissue rapidly transported to the laboratory. The time interval between surgery and the beginning of the experimental procedure did not exceed 60 min. Personal data and histopathological features are summarized in Table 1. Experimental procedures In principle, we followed the procedure used for the study of KYNA production in rat brain slices (Turski et al. 1989). Briefly, tissue derived from the human gliomas was sliced (base 1 × 1 mm) with a McIlwain chopper and preincubated for 10 rain at 37 °C in culture wells containing 1 ml oxygenated KRB in the presence ("regular KRB") or absence of sodium and with continuous supply of oxygen. Subsequently, the preincubation medium was removed and collected, and the tissue incubated for an additional two hours under the same conditions in 1 ml fresh buffer either in the absence of kynurenine or in buffer containing 40, 200 or 1000/IM kynurenine. The number of experimental conditions was dictated by the amount of available tumor tissue. Two or 3 wells were used for each data point. Following incubation, the wells were placed on ice, tissue was separated from the incubation buffer, and preincubation and incubation media were frozen and stored at - 70 °C until analysis. For KYNA determination, 200/~1 1 N HCI were added to each sample, and proteins were precipitated by boiling (10 min) and removed by subsequent centrifugation (8730 × g, 10 rain). The resulting supernatant was applied to a Dowex 50W (H + form; Sigma) cation-exchange column (0.5 × 2 cm). Subsequently, the column was

53 TABLE 1 PATIENTS AND HISTOPATHOLOGICALFEATURES Patient

Age (yrs)

Sex

Diagnosis

Site of tumor

A

32

M

Anaplasticastrocytoma

B

49

M

Astrocytoma

C D E F G

64 59 59 63 65

F M M M M

Anaplastieastrocytoma Astrocytoma Astroeytoma Anaplasticastrocytoma Astrocytoma

Frontal parietal cingular lobe, right hemisphere Fronto-temporo insular lobe, bordering ventricleleft hemisphere Parietal lobe, left hemisphere Parietal lobe, right hemisphere Parietal lobe, right hemisphere Frontal lobe, left hemisphere Temporal lobe, left hemisphere

H I J K L M N

54 71 54 39 51 67 69

M M M M F M M

Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma

Temporal lobe, left hemisphere Frontal lobe, right hemisphere Occipital lobe, right hemisphere Parietal lobe, left hemisphere Frontal lobe, left hemisphere Frontal lobe, left hemisphere Parietal lobe, left hemisphere

washed with 1 ml 0.1 N HCI and 1 ml of distilled water, and the fraction containing KYNA was eluted with 2 ml of water, lyophilized and resuspended in 240 #1 distilled water. Two hundred microliters were subjected to high performance liquid chromatography (HPLC), and KYNA was detected by UV absorption at 340 nm (Beckman 160 Absorbance Detector) as described previously (Turski et al. 1988, 1989; sensitivity limit: 2 pmol). Tissue slices were used for protein determination according to the method of Lowry et al. (1951). Further identification of the KYNA-like compound characterized by H P L C was accomplished using 4 different thin layer chromatography (TLC) systems. Tissue samples derived from gliomas B and C (astrocytomas) and glioma N (glioblastoma) were incubated separately with 1 mM kynurenine and their 2 h incubation media were collected and processed as described above. Following H P L C separation, samples and separate standard [3H]KYNA (Amersham) were concentrated and applied to TLC plates, and chromatograms were developed in one direction on cellulose or silica gel plates (Eastman Kodak). Subsequently, the plates were cut into 1-cm strips from the origin to the solvent front, eluted in distilled water and the powder was removed by centrifugation. The Rf values of [3H]KYNA were determined by liquid scintillation spectrometry, and the chemical identity of the corresponding sample strips was assessed by additional processing through the Dowex/HPLC separation procedure. Informed consent and statistics Informed consent was obtained from all patients prior to surgery. The statistical difference between astrocytomas and glioblastomas was evaluated using the Mann-Whitney U-test.

54 RESULTS The preincubation medium ("regular K R B " ) of 8 gliomas contained small concentrations of K Y N A , namely 1.5 + 0.7 pmol/mg protein, indicating the liberation of endogenous K Y N A into the medium. I n the remaining 6 gliomas, n o K Y N A could be detected, possibly because of the paucity of available tissue. A subsequent 2-h incubation period in the absence of kynurenine yielded trace a m o u n t s of K Y N A in the medium in 5 out of 11 gliomas. I n contrast, exposure to its bioprecursor kynurenine resulted in dose-dependent increases of K Y N A in the incubation medium in all specimens (Table 2). Notably, astrocytomas produced more K Y N A than glioblastomas at all 3 doses of kynurenine used; for the incubation with 200/~M and 1 m M kynurenine, the difference was statistically significant ( P < 0.01). No differences in K Y N A production were observed between tissues incubated in regular K R B and in sodium-free buffer (data not shown). The c o m p o u n d detected by H P L C was identified as K Y N A in all 4 T L C systems. TABLE 2 KYNA SYNTHESIS BY HUMAN GLIOMAS Tissue slices were incubated with kynurenine (40, 200 or 1000tiM) as described in the text. Data are the mean of 2 wells or the mean + SEM of 3 wells per kynurenine concentration and represent the KYNA content of the medium after 2 h incubation. KYNA production (pmol/2 h/rag protein) 40 gM

200/aM

1000/~M

n.d. n.d. n.d. n.d. n.d. 6.6 6.2

45.1 90.1 + 3.7 21.7 +_ 5.5 34.1 27.4 n.d. n.d.

85.1 339.7 63.5 + 8.9 88.5 91.2 n.d. n.d.

6.4

43.7 + 12.2

133.6 + 51.8

n.d. n.d. n.d. 3.6 +_ 1.1 3.0 + 0.7 2.3 + 0.6 5.0 +_0.6

7.0 +_ 2.6 + n.d. 14.2 11.3 + n.d. 16.7 +

25.7 +_ 10.6 + 7.7 + n.d. n.d. n.d. 43.6 i

3.5 + 0.6

10.4 + 2.5**

Astrocytoma Patient A Patient B Patient C Patient D Patient E Patient F Patient G Mean _+SEM of astrocytomas

Glioblastoma Patient H Patient I Patient J Patient K Patient L Patient M Patient N Mean +_SEM of glioblastomas

4.0 0.4 2.1 1.6

1.3 2.0 2.5

4.6

21.9 + 8.2**

n.d. = not done; tumor biopsies were too small to provide sufficienttissue for all kynurenineconcentrations. **P < 0.01 as compared to astrocytomas (Mann-Whitney U-test).

55 Thus, identical Rf values were obtained for standard [3H]KYNA and the KYNA-Iike substance isolated from the incubation media of gliomas B, C and N (Table 3).

DISCUSSION The present results demonstrate that human gliomas are capable of producing KYNA from its bioprecursor L-kynurenine. Although the limited availability of biopsy material precluded the proper assessment of dose-response relationships, the experimental data clearly demonstrated increased KYNA synthesis when kynurenine concentrations were raised up to 1 raM. Notably, the identity of the reaction product was verified by H P L C and by TLC analysis in four separate solvent systems. KYNA production by human gliomas appears to resemble the respective biosynthetic process for KYNA in rat brain tissue (Turski et al. 1989). Thus, it is possible to analyze in surgical specimens a composite process, consisting of cellular kynurenine uptake, intracellular transamination and subsequent efflux of KYNA into the extracellular space, with the experimental approach used for characterizing KYNA synthesis in rat brain slices. In contrast to the latter, however, KYNA production in the gliomas was not influenced by the presence of sodium in the incubation medium. This difference is possibly due to the virtual absence of neuronal elements in the human material since neurons possess sodium-dependent kynurenine uptake sites (Speciale and Schwarcz 1990), and since sodium-dependent neuronal activity can influence KYNA production in rat brain slices (Gramsbergen et al. 1988). Human gliomas produced substantially less KYNA than rat brain slices when normalized for tissue amount and bioprecursor concentration. The reason could lie in TABLE 3 IDENTIFICATION OF KYNA PRODUCTION BY HUMAN GLIOMA Comparison of standard [3H]KYNAand the KYNA-Iikesubstance isolated from incubation media of gliomas exposed to 1 mM kynurenine.See Methods for experimentaldetails. Two separate experimentswere performedwith astrocytomas C and D (experiment1) and glioblastoma N (experiment2), respectively. Data represent Rf values from 4 different thin-layer-chromatography(TLC) systems. TLC system

Cellulose plates 4~ sodium citrate Methanol/water/acetic acid (20:79:1) n-Propanol/2% ammonium acetate (1:1) Silica gel plate n-Butanol/methanol~water~ ammonium hydroxide(60:20:19:1)

Experiment 1

Experiment2

[3H]KYNA

GliomaC

GliomaD

[3H]KYNA GliomaN

0.42

0.42

0.42

0.45

0.44

0.73

0.73

0.74

0.73

0.74

0.87

0.86

0.87

0.83

0.82

0.65

0.65

0.64

0.61

0.61

56 the pathological nature of the excised surgical tissue which may contain only few kynurenine uptake sites or low kynurenine transaminase activity. The relatively modest output of K Y N A from human glioma compared to rat brain slices is certainly at odds with measurements of K Y N A content in normal human and rodent brain showing a more than 10-fold higher K Y N A concentration in the human tissue (Carl~t et al. 1988; Turski et al. 1988). The relative preponderence of K Y N A production in astrocytomas as compared to the less differentiated glioblastomas is in accordance not only with the proven ability of (rodent) astrocytes to rapidly accumulate kynurenine in a sodium-independent fashion (Speciale et al. 1989) but more generally with our current understanding of kynurenine neurobiology. Thus, at least two of the enzymes involved in the metabolism of K Y N A ' s excitotoxic relative, quinolinic acid, are localized in astrocytes in both rodent and human brain (Schwarcz et al. 1989). Moreover, an increased production of K Y N A from kynurenine has been observed in lesioned, neuron-depleted brain tissue at a time of pronounced astrocytic proliferation (Turski et al. 1989). It is difficult to evaluate the possible functional significance of the present findings. Kynurenine concentrations in the mammalian brain are in the low micromolar range (Joseph 1978), so that glial tumors can be expected to produce K Y N A concentrations in the high nanomolar range. In extrapolation from work on rodent brains, in particular recent revelations concerning the high potency of K Y N A as an antagonist at the glycine-regulated channel associated with the N-methyl-D-aspartate receptor (Kessler et al. 1989), it is therefore conceivable that tumor-derived K Y N A can interfere with central excitatory amino acid receptor function. It remains to be elaborated if and to what extent glial tumors indeed produce K Y N A in situ and how glioma-derived K Y N A may play a role in pathophysiological conditions.

ACKNOWLEDGEMENTS This work was supported in part by U S P H S grant NS 16102 and Italian National Research Council Grant 87.00041.04. We gratefully appreciate the help of Dr. V. Monte and Ms. L. Besozzi, and the expert secretarial assistance of Ms. Joyce Burgess. REFERENCES Andinr, P., A. Lehmann, K. Ellrrn, E. Wennberg, I. Kjellmer, T. Nielsen and H. Hagberg (1988) The excitatory amino acid antagonist kynurenic acid administered after hypoxic ischemia in neonatal rats offers neuroprotection. Neurosci. Lett., 90: 208-212. Carla, V., G. Lombardi,M. Beni,P. Russi, G. Moneti and F. Moroni (1988) Identificationand measurement of kynurenic acid in the rat brain and other organs. Anal. Biochem., 169: 89-94. Gramsbergen, J.B.P., W.A. Turski and R. Sehwaxcz (1988) Neuronal activity affects kynurenic acid production in rat brain slices. Soc. Neurosci. Abstr., 14: 479.12. Joseph, M. H. (1978) Determination of kynurenine by a simplegas-liquid chromatographicmethod applicable to urine, plasma, brain and eerebrospinal fluid. J. Chromatogr., 146: 33-41. Kessler, M., T. Terramani, G. Lynch and M. Baudry (1989) A glycine site associated with N-methylD-aspartic acid receptors: characterization and identification of a new class of antagonists. J. Neurochem., 52: 1319-1328.

57 Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 263-275. Minatogawa, Y., T. Noguchi and R. Kido (1974) Kynurenine pyruvate transaminase in rat brain. J. Neurochem., 23: 271-272. Nakamura, M., E. Okuno, W. O. Whetsell and R. Schwarcz (1988) Kynurenine aminotransferase in human brain. Soc. Neurosci. Abstr., 14: 479.10. Perkins, M.N. and T.W. Stone (1982) An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid. Brain Res., 247: 183-187. Schwarcz, R. and B. Meldrum (1985) Excitatory amino acid antagonists provide a therapeutic approach to neurological disorders. Lancet, 2: 140-143. Schwarcz, R., A.C. Foster, E.D. French, W.O. Whetsell and C. K6hler (1984) Excitotoxic models for neurodegenerative disorders. Life Sci., 35: 19-32. Schwarcz, R., C. Speeiale and W.A. Turski (1989) Kynurenines, glia, and the pathogenesis of neurodegenerative disorders. In: Calne, D. B., Comi, G., Crippa, D., I-Iorowski, R. and Trabucchi, M. (Eds.), Parkinson's Disease and Aging, Raven Press, New York, pp. 97-105. Simon, R. P. (1988) Focal ischemia and excitatory amino acid antagonists. In: Cavalheiro, E.A., Lehmann, J. and Turski, L. (Eds.), Frontiers in Excitatory Amino Acid Research, Alan Liss, New York, pp. 639-644. Speciale, C. and R. Schwarcz (1990) Uptake ofkynurenine into rat brain slices. J. Neurochem., 54:156-163. Speciale, C., K. Hares, R. Schwartz and N. Brookes (1989) High-affinity uptake of L-kynurenine by a Na + -independent transporter of neutral amino acids in astrocytes. J. Neurosci., 9: 2066-2072. Stone, T. W. and J. H. Connick (1985) Quinolinic acid and other kynurenines in the central nervous system. Neuroscience, 15: 597-617. Turski, W.A., M.N. Nakamura, W.P. Todd, B.K. Carpenter, W.O. Whetsell and R. Schwarcz (1988) Identification and quantification of kynurenic acid in human brain tissue. Brain Res., 454: 164-169. Turski, W.A., J.B.P. Gramsbergen, H. Traltler and R. Schwarcz (1989) Rat brain slices produce and liberate kynurenic acid upon exposure to L-kynurenine. J. Neurochem., 52: 1629-1636.