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Toxicity of sulphur-containing compounds to neuronal cell lines
JOURNAL OF THE
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 129 (Suppl.) (1995) 107-108
Toxicity of sulphur-containing compounds to neuronal cell lines A . R . P e a n ", R . B . P a r s o n s
", R . H . W a r i n g
a, * ,
A . C . W i l l i a m s b, D . B . R a m s d e n
c
School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK b Department of Clinical Neurology, Queen Elizabeth Hospital, Edgbaston, Birmingham, B15 2TH, UK c Department of Medicine, The University of Birmingham, Edgbaston, Birmingham, B15 277] UK Accepted 21 February 1995
Abstract
Cysteine and its metabolites cysteine sulphinic acid (CSA) and taurine (TA) were shown to be toxic to human and rat neuronal cell lines. However, the mechanisms of action of CSA and TA appeared to be different. As MND/ALS patients have high plasma and CSF cysteine levels, they may have increased levels of neurotoxins in vivo, since cysteine was more toxic than CSA in the assay system used. Cysteic acid, homocysteic acid, BMAA and BOAA were also toxic to the cell lines used.
Keywords: MND/ALS; Cysteine; Neurones; Toxicity; Rat neuronal cell line; Human neuroblastoma ceils; Cysteic acid; Cysteine; Cysteine sulphinic acid; Taurine; Homocysteic acid; /3-N-Methylamino-L-alanine; /3-N-Oxalamino-L-alanine; Sulphate; Sporadic amyotrophic lateral sclerosis
1. Introduction
2. Materials and m e t h o d s
We have previously shown that plasma levels of cysteine are elevated in motor neurone disease ( M N D / A L S ) , and that levels of sulphate are reduced (Heaficld et al., 1990). Cysteine is an excitotoxin which acts on the N M D A subtype of glutamate receptor, finally leading to cell death (Olney et al., 1990). Other sulphur-containing metabolites of cysteine such as cysteine sulphinic acid (CSA) have also been shown to be neurotoxic (Klingman and Choi, 1989; Griffiths, 1990). Cysteine is metabolised in vivo by oxidative degradation to inorganic sulphate, via CSA. The formation of CSA from cysteine is carried out by the enzyme cysteine oxidase, which has been shown in vitro to be of reduced activity in M N D / A L S patients (Foster et al., 1991). Taurine (TA) can either be formed from CSA or from cysteamine. As M N D / A L S patients may have altered CSA and T A levels, these compounds, with other known neurotoxins, were investigated for toxicity to neuronal cell cultures.
Cysteine, glutamic acid, taurine and homocysteic acid were all supplied by Sigma Chemicals, cysteine sulphinic acid and cysteic acid were from Alldrich. B M A A and B O A A were a gift from Dr. P.B. Nunn. Rat neuronal cell lines (B50 and B65) and SKNSH human neuroblastoma cells were maintained in culture medium. The rat cell lines were grown in RMP1 1640 culture medium (Gibco BRL, Uxbridge, Middlesex, UK) supplemented with 10% heat inactivated foetal calf serum, L-glutamine (2 mM) penicillin and streptomycin (1000 and 500 u n i t s / m l respectively). The SKNSH cells were maintained in D M E M culture medium (Gibco) also supplemented as for the rat cell lines, and including pyruvic acid (1 mM). Cells were grown in cell culture flasks in a humidified 95% 0 2 / 5 % CO 2 atmosphere at 37 ° C. Sulphur-containing compounds and neurotoxins were added to culture media at the appropriate concentrations and the resulting solutions corrected for pH. Quin-2 was added to a final concentration of 20 p~M to certain experiments. Confluent cells grown for 3 days had the growth medium removed and replaced with test medium. At the end of the test period, the test medium was removed and stored at - 20 ° C until it
* Corresponding author. Fax: ( + 44-121 ) 414 3982. 0022-510X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 2 2 - 5 1 0 X ( 9 5 ) 0 0 0 7 8 - X
108
A.R. Pean et al. /Journal of the Neurological Sciences 129 (SuppL) (1995) 107-108 ~.00
cysfelne
3oo
_~ zoo
however toxic at a range of 0.5-2.0 raM. T h e calcium chelator Quin-2 blocked L D H release in I350 cells incubated with taurine and B O A A and in B65 cells incubated with taurine and glutamate.
o ~00' tystelne
g
0 5O
100
120
I~0
160
IBO
200
[ Substrote ] ~M
Fig. 1. Percentage increase in SKNSH cell death above controls after 24 h incubation times. A, cysteine: m, cysteine sulphinic acid; o, cysteic acid. Results were carried out in triplicate; the mean is plotted, the ranges being + 10%. was assayed for L D H activity. T h e release of L D H from cells was used as a measure of cell toxicity and death and was estimated by following the decrease in absorbance of N A D H at 340 nm, following the m e t h o d of Mold6us et al. (1978). Control samples without additions to the m e d i u m but incubated for the same time period were taken as b a c k g r o u n d cell death. Each result was carried out in triplicate; means were plotted (Fig. 1), the ranges being _+ 10%.
3. Results
As can be seen from Fig. 1, cysteine, cysteine sulphinic acid (CSA), cysteic acid (CA) all show toxicity to the S K N S H h u m a n n e u r o b l a s t o m a cell culture after 24 h incubation. All three c o m p o u n d s show a biphasic response, with maximal toxicity at low concentrations (10 /~M for cysteine and cysteine sulphinic acid) and then a second peak a r o u n d 200 # M . Homocysteic acid ( H C A ) was also toxic to S K N S H cells, with maximal L D H release at 25 # M ; toxicity at 100 and 200 p~M was equal to control values. Using B50 cells, d o s e - d e p e n d e n t responses at 1 h incubation were seen for C S A (0.1-2 raM), taurine (TA) ( 1 - 2 raM), B M A A and B O A A (0.1-1.0 mM). With increasing incubation time, no differences were seen in test and control samples, suggesting that the cells had substantial repair capacity. The B65 cell line showed d o s e - d e p e n d e n t increases in L D H release with CSA, T A and glutamic acid; the maximal toxicity for C S A was seen on 5h incubation (0.1-2 m M ) almost identical with that seen for glutamate; maximal toxicity for taurine was seen with an incubation time of 24 h. Time profiles for the S K N S H cells, where glutamate showed toxicity over the 0 . 0 1 - 2 m M range of concentrations, were different for C S A and TA. Both were
4. D i s c u s s i o n
These results demonstrate the toxicity of TA, CA, C S A and H C A , all metabolites or precursors of cysteine, to neuronal cell lines and show that they are c o m p a r a b l e with known neurotoxins such as B M A A and B O A A . They a p p e a r to differ from each other in their mechanisms of action a n d / o r receptor specificity as their toxicity profiles and response to Quin-2 are not the same. However, cysteine and its oxidised p r o d u c t cysteine sulphinic acid have the same toxicity response profile, and may therefore have the same m e c h a n i s m for causing cell damage. Cysteine appears to be about 10-fold more toxic than its metabolite. As patients with M N D / A L S have high plasma cysteine concentrations and r e d u c e d capacity for its oxidation to the less toxic products, they may be more at risk of chronic n e u r o n a l damage. F u r t h e r work is n e e d e d to assess the implications of differential neuronal toxicity and n e u r o m o d u lation in M N D / A L S patients,
Acknowledgements
This work was supported by a grant from the M o t o r N e u r o n e Disease Association. We thank Dr. P. B. N u n n for his gift of B O A A and B M A A .
References
Foster, H., Young, T.W., Waring, R.H. and Elias, E. (1991) Cysteine oxidase activity and properties in rat and human liver cytosol. Hepatology, 14: 222A. Griffiths, R. (1990) Cysteine sulphinate (CSA) as an excitatory amino acid transmitter candidate in the mammalian central nervous system. Prog. Neurobiol., 35: 313-323. Heafield, M.T.E., Fearn, S., Steventon, G.B., Waring, R.H., Williams, A.C. and Sturman, S.G. (1990) Plasma cysteine and sulphate levels in motor neurone, Parkinson's and Alzheimer's disease. Neurosci. Lett., 110: 216-220. Klingman, J.G. and Choi, D.W. (1989) Toxicity of sulphur-containing amino acids on cultured cortical neurones. Neurology, 39: 2425. Mold~us, P., Hogberg, J. and Orrenius, S. (1978) Isolation and uses of liver cells. Methods Enzymol., 52: 60-71. Olney, J.W., Zorumski, C., Price, M.T. and Labruyere, J. (1990) L-Cysteine, a bicarbonate-sensitive endogenous excitotoxin. Science, 248: 596-599.
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 129 (Suppl.) (1995) 107-108
Toxicity of sulphur-containing compounds to neuronal cell lines A . R . P e a n ", R . B . P a r s o n s
", R . H . W a r i n g
a, * ,
A . C . W i l l i a m s b, D . B . R a m s d e n
c
School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK b Department of Clinical Neurology, Queen Elizabeth Hospital, Edgbaston, Birmingham, B15 2TH, UK c Department of Medicine, The University of Birmingham, Edgbaston, Birmingham, B15 277] UK Accepted 21 February 1995
Abstract
Cysteine and its metabolites cysteine sulphinic acid (CSA) and taurine (TA) were shown to be toxic to human and rat neuronal cell lines. However, the mechanisms of action of CSA and TA appeared to be different. As MND/ALS patients have high plasma and CSF cysteine levels, they may have increased levels of neurotoxins in vivo, since cysteine was more toxic than CSA in the assay system used. Cysteic acid, homocysteic acid, BMAA and BOAA were also toxic to the cell lines used.
Keywords: MND/ALS; Cysteine; Neurones; Toxicity; Rat neuronal cell line; Human neuroblastoma ceils; Cysteic acid; Cysteine; Cysteine sulphinic acid; Taurine; Homocysteic acid; /3-N-Methylamino-L-alanine; /3-N-Oxalamino-L-alanine; Sulphate; Sporadic amyotrophic lateral sclerosis
1. Introduction
2. Materials and m e t h o d s
We have previously shown that plasma levels of cysteine are elevated in motor neurone disease ( M N D / A L S ) , and that levels of sulphate are reduced (Heaficld et al., 1990). Cysteine is an excitotoxin which acts on the N M D A subtype of glutamate receptor, finally leading to cell death (Olney et al., 1990). Other sulphur-containing metabolites of cysteine such as cysteine sulphinic acid (CSA) have also been shown to be neurotoxic (Klingman and Choi, 1989; Griffiths, 1990). Cysteine is metabolised in vivo by oxidative degradation to inorganic sulphate, via CSA. The formation of CSA from cysteine is carried out by the enzyme cysteine oxidase, which has been shown in vitro to be of reduced activity in M N D / A L S patients (Foster et al., 1991). Taurine (TA) can either be formed from CSA or from cysteamine. As M N D / A L S patients may have altered CSA and T A levels, these compounds, with other known neurotoxins, were investigated for toxicity to neuronal cell cultures.
Cysteine, glutamic acid, taurine and homocysteic acid were all supplied by Sigma Chemicals, cysteine sulphinic acid and cysteic acid were from Alldrich. B M A A and B O A A were a gift from Dr. P.B. Nunn. Rat neuronal cell lines (B50 and B65) and SKNSH human neuroblastoma cells were maintained in culture medium. The rat cell lines were grown in RMP1 1640 culture medium (Gibco BRL, Uxbridge, Middlesex, UK) supplemented with 10% heat inactivated foetal calf serum, L-glutamine (2 mM) penicillin and streptomycin (1000 and 500 u n i t s / m l respectively). The SKNSH cells were maintained in D M E M culture medium (Gibco) also supplemented as for the rat cell lines, and including pyruvic acid (1 mM). Cells were grown in cell culture flasks in a humidified 95% 0 2 / 5 % CO 2 atmosphere at 37 ° C. Sulphur-containing compounds and neurotoxins were added to culture media at the appropriate concentrations and the resulting solutions corrected for pH. Quin-2 was added to a final concentration of 20 p~M to certain experiments. Confluent cells grown for 3 days had the growth medium removed and replaced with test medium. At the end of the test period, the test medium was removed and stored at - 20 ° C until it
* Corresponding author. Fax: ( + 44-121 ) 414 3982. 0022-510X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 2 2 - 5 1 0 X ( 9 5 ) 0 0 0 7 8 - X
108
A.R. Pean et al. /Journal of the Neurological Sciences 129 (SuppL) (1995) 107-108 ~.00
cysfelne
3oo
_~ zoo
however toxic at a range of 0.5-2.0 raM. T h e calcium chelator Quin-2 blocked L D H release in I350 cells incubated with taurine and B O A A and in B65 cells incubated with taurine and glutamate.
o ~00' tystelne
g
0 5O
100
120
I~0
160
IBO
200
[ Substrote ] ~M
Fig. 1. Percentage increase in SKNSH cell death above controls after 24 h incubation times. A, cysteine: m, cysteine sulphinic acid; o, cysteic acid. Results were carried out in triplicate; the mean is plotted, the ranges being + 10%. was assayed for L D H activity. T h e release of L D H from cells was used as a measure of cell toxicity and death and was estimated by following the decrease in absorbance of N A D H at 340 nm, following the m e t h o d of Mold6us et al. (1978). Control samples without additions to the m e d i u m but incubated for the same time period were taken as b a c k g r o u n d cell death. Each result was carried out in triplicate; means were plotted (Fig. 1), the ranges being _+ 10%.
3. Results
As can be seen from Fig. 1, cysteine, cysteine sulphinic acid (CSA), cysteic acid (CA) all show toxicity to the S K N S H h u m a n n e u r o b l a s t o m a cell culture after 24 h incubation. All three c o m p o u n d s show a biphasic response, with maximal toxicity at low concentrations (10 /~M for cysteine and cysteine sulphinic acid) and then a second peak a r o u n d 200 # M . Homocysteic acid ( H C A ) was also toxic to S K N S H cells, with maximal L D H release at 25 # M ; toxicity at 100 and 200 p~M was equal to control values. Using B50 cells, d o s e - d e p e n d e n t responses at 1 h incubation were seen for C S A (0.1-2 raM), taurine (TA) ( 1 - 2 raM), B M A A and B O A A (0.1-1.0 mM). With increasing incubation time, no differences were seen in test and control samples, suggesting that the cells had substantial repair capacity. The B65 cell line showed d o s e - d e p e n d e n t increases in L D H release with CSA, T A and glutamic acid; the maximal toxicity for C S A was seen on 5h incubation (0.1-2 m M ) almost identical with that seen for glutamate; maximal toxicity for taurine was seen with an incubation time of 24 h. Time profiles for the S K N S H cells, where glutamate showed toxicity over the 0 . 0 1 - 2 m M range of concentrations, were different for C S A and TA. Both were
4. D i s c u s s i o n
These results demonstrate the toxicity of TA, CA, C S A and H C A , all metabolites or precursors of cysteine, to neuronal cell lines and show that they are c o m p a r a b l e with known neurotoxins such as B M A A and B O A A . They a p p e a r to differ from each other in their mechanisms of action a n d / o r receptor specificity as their toxicity profiles and response to Quin-2 are not the same. However, cysteine and its oxidised p r o d u c t cysteine sulphinic acid have the same toxicity response profile, and may therefore have the same m e c h a n i s m for causing cell damage. Cysteine appears to be about 10-fold more toxic than its metabolite. As patients with M N D / A L S have high plasma cysteine concentrations and r e d u c e d capacity for its oxidation to the less toxic products, they may be more at risk of chronic n e u r o n a l damage. F u r t h e r work is n e e d e d to assess the implications of differential neuronal toxicity and n e u r o m o d u lation in M N D / A L S patients,
Acknowledgements
This work was supported by a grant from the M o t o r N e u r o n e Disease Association. We thank Dr. P. B. N u n n for his gift of B O A A and B M A A .
References
Foster, H., Young, T.W., Waring, R.H. and Elias, E. (1991) Cysteine oxidase activity and properties in rat and human liver cytosol. Hepatology, 14: 222A. Griffiths, R. (1990) Cysteine sulphinate (CSA) as an excitatory amino acid transmitter candidate in the mammalian central nervous system. Prog. Neurobiol., 35: 313-323. Heafield, M.T.E., Fearn, S., Steventon, G.B., Waring, R.H., Williams, A.C. and Sturman, S.G. (1990) Plasma cysteine and sulphate levels in motor neurone, Parkinson's and Alzheimer's disease. Neurosci. Lett., 110: 216-220. Klingman, J.G. and Choi, D.W. (1989) Toxicity of sulphur-containing amino acids on cultured cortical neurones. Neurology, 39: 2425. Mold~us, P., Hogberg, J. and Orrenius, S. (1978) Isolation and uses of liver cells. Methods Enzymol., 52: 60-71. Olney, J.W., Zorumski, C., Price, M.T. and Labruyere, J. (1990) L-Cysteine, a bicarbonate-sensitive endogenous excitotoxin. Science, 248: 596-599.