Toxicology in Vitro 16 (2002) 481–483

www.elsevier.com/locate/toxinvit

Cysteine dioxygenase: modulation of expression in human cell lines

by cytokines and control of sulphate production
L.J. Wilkinson, R.H. Waring*
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK

Abstract
Cysteine dioxygenase (CDO) is the initial and rate-limiting enzyme involved in the oxidative degradation of cysteine to inorganic
sulphate. It is believed to be the major source of sulphate in vivo. Inflammatory conditions such as rheumatoid arthritis have been
linked with high plasma cysteine:sulphate ratios in patients. The cytokines tumour necrosis factor-a (TNF-a) and transforming
growth factor-b (TGF-b) have been shown to inhibit the expression of CDO in neuronal (TE671) and hepatic (Chang) human cell
lines at nanomolar concentrations. Cytokine release may therefore modulate sulphate production and hence regulate formation of
sulphated biocomponents. # 2002 Published by Elsevier Science Ltd.
Keywords: Cysteine dioxygenase; Sulfate; Cytokine; TNF-a; Human cell lines

1. Introduction cytokines are released in inflammation, the present study

Cysteine dioxygenase (CDO) is a cytosolic enzyme,
found primarily in liver (Parsons et al., 1998b) and brain
(Parsons et al., 1998a), with some activity in kidney, heart
and thyroid (Parsons et al., 2001). The enzyme carries
out the conversion of cysteine to cysteine sulphinic acid,
the initial step in a pathway which leads to the forma-
tion of inorganic sulphate. The CDO-catalysed step is
believed to be rate-limiting and this metabolic route is
responsible for the production of the majority of sul-
phate in vivo (Sörbo and Ewetz, 1965), as absorption of
sulphate across the gastrointestinal tract is relatively
inefficient in man (5–25%) (Florin et al., 1991). Other
pathways for sulphate formation exist, but are of much
less importance (Sörbo and Ewetz, 1965). Various con-
ditions with an autoimmune or inflammatory compo-
nent, such as rheumatoid arthritis (RA) (Bradley et al.,
1994), systemic lupus erythematosus (SLE) (Gordon et
al., 1992) and primary biliary cirrhosis (Davies et al.,
1995) have been shown to be linked with high plasma
levels of cysteine and low plasma concentrations of
inorganic sulphate in patients with these diseases. As
Abbreviations: CDO, cysteine dioxygenase; DMEM, Dulbecco’s
modified Eagle’s medium; EMEM, Eagle’s modified Eagle’s medium;
HIFBS, heat-inactivated foetal bovine serum; LDH, lactate dehy-
drogenase; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus;
TGF-b, transforming growth factor-b; TNF-a, tumour necrosis factor-a
* Corresponding author. Tel.: +44-121-414-5421; fax: +44-121-
414-3982.
0887-2333/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0887-2333(02)00031-0
was carried out to determine whether they could alter
the expression of CDO in human hepatic and neuronal
cell lines.
2. Materials and methods
2.1. In vitro culture of human cell lines
The cell lines were obtained from the European Col-
lection of Animal Cell Cultures (Porton Down, UK) as
frozen ampoules. All tissue culture reagents were pur-
chased from Sigma Chemical Company (Poole, UK).
TE671 cells (human medulloblastoma) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM), supple-
mented with 10% heat-inactivated foetal bovine serum
(HIFBS), 2 mmol/l glutamine, 100 U/ml penicillin and
100 mg/ml streptomycin. Chang human hepatocytes
were cultured in Eagle’s modified Eagle’s medium
(EMEM), with the above supplements and the addition
of non-essential amino acids (5%). Cells were main-
tained in a humidified tissue culture incubator at 37  C,
5% CO2. Medium was changed every 2 days and cells
were subcultured 1:5 when confluent.
2.2. Cytokine administration
Cells were plated out into six-well tissue culture plates
at a density of 1106 cells/ml. They were cultured
482 L.J. Wilkinson, R.H. Waring / Toxicology in Vitro 16 (2002) 481–483

overnight, after which the medium was removed and

replaced with medium containing tumour necrosis fac-
tor-a (TNF-a; 0, 0.01, 0.05, 0.1, 0.25, 0.5 ng/ml) or
transforming growth factor-b (TGF-b; 0, 1, 2.5, 5, 7.5,
10 ng/ml) for 24 or 48 h. The medium was retained for
lactate dehydrogenase (LDH) analysis using a Cytotox
961 cytotoxicity assay kit (Promega, Southampton,
UK), according to the manufacturer’s instructions.
Cells were lysed in 200 ml lysis buffer (1% Triton-
X100 in Tris-buffered saline) to obtain protein for SDS–
PAGE/Western blotting. Protein concentrations of cel-
lular lysates were determined according to a modified
version of the method of Bradford (1976).
Denatured samples (20 mg) were electrophoresed as
previously described (Qusti et al., 2000) and gels were
stained after electrophoresis for protein with Brilliant
Blue stain. Alternatively, proteins were transferred to
nitrocellulose blotting membrane. The membrane was
Fig. 1. Western blot of Chang cells dosed with cysteine, TNF-a or
probed initially with HCDO antibody (1:50, The Bind-
TGF-b for 24 h.
ing Site, Birmingham, UK) raised in sheep, followed by
anti-sheep IgG (1:500, Dako Ltd, Ely, Cambridgshire,
Table 1
UK) conjugated to horseradish peroxidase. Detection of
Effect of treatment with TNF-a on CDO expression in Chang and
the specific protein was carried out via enhanced chemilu- TE671 cells
minescence, using ECLTM Western blotting detection
reagents (Amersham Pharmacia Biotech, Buckingham- Expression (% of 0 ng/ml expression)
shire, UK). Films were analysed by the Kodak digital Chang cell line TE671 cell line
science 1D programme. Band intensities were measured
TNF-a (ng/ml) 24 h 48 h 24 h 48 h
via densitometry and levels of CDO expression were cal-
culated as percentages of intensity of the control band. 0 100.0 100.0 100.0 100.0
All experiments were conducted in triplicate to ensure 0.01 44.314.3 76.415.4 60.85.9 74.28.8
0.05 24.78.6 61.216.7 44.115.8 73.315.4
reproducibility.
0.10 17.55.7 55.112.2 41.711.0 61.817.5
0.25 15.56.3 42.99.8 27.09.6 57.56.6
0.50 14.83.1 38.411.5 19.97.4 41.312.2
3. Results

A band of approximately 70 kDa was observed in all

Table 2
treatment groups and controls. This was in agreement Effect of treatment with TGF-b on CDO expression in Chang and
with that reported previously (Qusti et al., 2000) and is the TE671 cells
main form of CDO detected with this antibody. Treat-
Expression (% of 0 ng/ml expression)
ment of Chang cells with cysteine for 24 h resulted in an
up-regulation of CDO expression by 353% at 400 mm Chang cell line TE671 cell line
(Fig. 1a). In TE671 cells the expression was up-regulated
TGF-b (ng/ml) 24 h 48 h 24 h 48 h
by 120%. Treatment of cells with TNF-a was followed by
a down-regulation of CDO expression in both cell types 0 100.0 100.0 100.0 100.0
(Table 1) which was maximal at 24 h. In Chang cells, the 1.0 54.88.5 60.612.1 71.613.6 80.814.3
2.5 52.35.3 65.17.9 70.58.5 69.010.1
0.5 ng/ml dose led to a reduction in CDO expression of
5.0 34.711.4 58.710.1 54.16.2 64.28.6
greater than 85% (Fig. 1b). This level also gave more than 7.5 19.59.9 57.99.6 42.87.4 55.611.3
80% reduction in expression for TE671 cells. No corre- 10.0 18.34.6 59.115.7 32.410.8 48.715.5
sponding increase in cell death was observed.
TGF-b was also shown to down-regulate CDO
expression (Table 2) with maximal effects again
observed at 24 h. A greater than 80% reduction in CDO 4. Discussion
expression was seen in Chang cells at 24 h, compared to
the control cells (Fig. 1c). The expression of the protein The increase in CDO expression on treatment with
also decreased in TE671 cells, although to a lesser extent cysteine has previously been reported for the TE-671
( 68%). Again no increase in cell death was observed. cell line (Qusti et al., 2000). In vivo experiments in
 L.J. Wilkinson, R.H. Waring / Toxicology in Vitro 16 (2002) 481–483
which rats were dosed with cysteine also increased
hepatic CDO (Kohashi et al., 1978; Parsons et al.,
1998b) and our in vitro results with Chang cells are in
agreement with these findings. Treatment with both
TNF-a and TGF-b gave rise to a reduction in the
expression of CDO in both cell lines studied. This was
an unexpected finding since these cytokines are thought
to have opposing effects on their cellular targets. The
down-regulation of the expression of CDO by cytokines
in vitro provides a possible mechanism to explain the
clinical findings of low plasma sulphate in inflammatory
conditions. Patients with RA have also been shown to
have reduced sulphation of synovial fluid and proteins
and glycosaminoglycans within the joint itself, which
would reduce the protective and lubricant effects of
these biocomponents (Bradley et al., 1994). Continued
cytokine release would gradually worsen the condition.
The finding that cytokines can affect sulphate produc-
tion is of importance in furthering the understanding of
chronic inflammatory disease processes; it is noteworthy
that antibodies to TNF-a are currently successfully used
as therapy for rheumatoid arthritis (Barrera et al., 2001;
Koopman, 2001).
Acknowledgements
We would like to thank Autism Unravelled and the
Autism Research Trust for supporting this work.
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