Oxidation and Generation of Hydrogen Peroxide by Thiol
Compounds in Commonly Used Cell Culture Media
Lee Hua Long and Barry Halliwell
1
Department of Biochemistry, Faculty of Medicine, National University of Singapore,
Medical Drive, MD7 #03-15, 10 Kent Ridge Crescent, Singapore 119260
Received July 30, 2001
Many studies have examined the effects of thiol com-
pounds upon cells in culture (e.g., upon signal trans-
duction and regulation of gene expression), but few
have considered how thiols can interact with cell cul-
ture media. A wide range of thiols (cysteine, GSH,
N-acetylcysteine, -glutamylcysteine, cysteinylglycine,
cysteamine, homocysteine) were found to interact
with three commonly used cell culture media (RPMI,
MEM, DMEM) to generate hydrogen peroxide with
complex concentration-dependencies. Thiols added to
these media rapidly disappeared, although less H
2
O
2
was generated on a molar basis than the amount of
thiol lost. Studies on cellular effects of thiols, espe-
cially those on redox regulation of gene expression or
protein function, need to take into account that thiols
are rapidly lost, and that their oxidation generates
H
2
O
2
, which can have multiple concentration– depen-
dent effects on cell metabolism. © 2001 Academic Press
Key Words: hydrogen peroxide; GSH; thiol; cysteine;
cysteamine; cell culture; homocysteine; autoxidation.
Compounds containing the thiol (-SH) group can
scavenge a wide range of reactive oxygen, chlorine, and
nitrogen species, including hydroxyl radical (OH
•
), hy-
drogen peroxide (H
2
O
2
), peroxyl radicals (RO
2
•
), per-
oxynitrite (ONOO
-
), and hypochlorous acid HOC1 (1–
7). Hence thiols are widely regarded as antioxidants;
indeed the most common intracellular thiol, reduced
glutathione (GSH) is a major contributor to cellular
antioxidant defences (8, 9) and N-acetylcysteine is
widely used as an antioxidant in vivo and in cell cul-
ture studies (5, 10 –12). Nevertheless, injurious effects
of certain thiols have been reported in several cell
culture studies. For example, cysteamine caused dam-
age to cultured astroglia (13), cysteamine, and N-acetyl-
cysteine caused DNA strand breaks in phagocytes (14),
cysteine was toxic to isolated rat hepatocytes (15), and
homocysteine accelerated senescence in cultured endo-
thelial cells (16). The toxicities of cysteine (15) and
cysteamine (17) to isolated cells have been suggested to
involve H
2
O
2
. Even GSH has been reported to damage
cells containing -glutamyl transpeptidase, by the
generation of H
2
O
2
(18, 19). The mutagenicity of cys-
teine and cysteinylglycine to Escherichia coli also ap-
peared to involve H
2
O
2
, in that it could be blocked by
catalase (20).
However, a factor not taken into account in most of
these studies is the reaction of thiols with cell culture
media. For example, we have recently shown that
ascorbate (21) and several polyphenolic compounds
(22) undergo rapid chemical reactions with commonly
used cell culture media to generate H
2
O
2
, and that this
H
2
O
2
production could account for many or all of the
reported effects of these compounds on cells in culture.
We have examined the reaction of several thiol com-
pounds with commonly used cell culture media to see if
a similar effect could occur. The present paper reports
our findings.
MATERIALS AND METHODS
Reagents. Dulbecco’s modified Eagle medium (DMEM) contain-
ing 5% fetal bovine serum and RPM1-1640 cell culture media were
obtained from Hyclone (Irvine, CA) and minimal essential medium
(MEM) was from Gibco, BRL (Gaithersburg, MD).
The thiols and DTNB were obtained from Sigma Chemicals Pte
Ltd., Singapore. All the thiols except cysteine were dissolved in
deionized water to a concentration of 10 mM immediately before use,
the pH was adjusted to 7.4, and the thiols were added to the culture
media to give the final concentration stated. For cysteine, 1 mM of
cysteine was prepared directly in the culture media and the pH was
adjusted to 7.4. Catalase was Sigma type C40 (specific activity 2500
units/mg protein). Tris base was from Research Organics (Cleveland,
Ohio) and EDTA from BDH Chemicals (Poole, UK).
Measurement of H
2
O
2
. A Hansatech oxygen electrode (Hansa-
tech, UK) was used (23, 24). The electrode was stabilized for 30 min
with 1.5 ml of air-saturated phosphate-buffered saline, pH 7.4 in the
chamber. The buffer was then replaced by 1.5 ml of culture medium
containing the thiols as stated and the recorder pen adjusted to a
position approximately 50% of full-scale deflection. One hundred
microliters of catalase solution (containing 1000 units) in phosphate-
1
To whom correspondence should be addressed. Fax: (+65) 779
1453. E-mail bchbh@nus.edu.sg.
Biochemical and Biophysical Research Communications 286, 991–994 (2001)
doi:10.1006/bbrc.2001.5514, available online at http://www.idealibrary.com on
991 0006-291X/01 $35.00
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.