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Effect of culture conditions on glutathione content in A549 cells
Vol. 114, No. 2, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
July 29, 1983
Pages
737-742
EFFECTSOF CULTURE CONDITIONSONGLUTATHIONE CONTENT IN A549 CELLS Gloria B. Post, Douglas A. Keller,
Karen A. Connor, and D. B. Mensel
Departments of Pharmacology and Medicine, Comprehensive Cancer Center Duke University Medical Center Durham, North Carolina 27710 Received June
17,
1983
Summarv: The effects of varying culture conditions on glutathione content in A549 (human type II lung tumor derived) cells were examined. Parameters studied were growth time, serum concentration, and the presence or absence of a mixture of insulin, transferrin, and selenous acid. Glutathione content When cells were grown with serum, increased with serum concentration. glutathione increased sharply 24 hours after passage and decreased thereafter. Insulin, transferrin, and selenous acid had little effect on cell growth or glutathione content. Replacement of media with fresh media containing 10% serum did not prevent the growth dependent decrease in glutathione. These results demonstrate that glutathione content in A549 cells is strongly affected by culture conditions.
The tripeptide .
intermediates destroy
glutathione
in several ways
free radicals
metabolites
affected
by the cell's
cultured
cell
their
of a particular
Cultured
cells
maintained
conditions
cell
have not
metabolism and toxicity
lines
(4,5,6),
usually
acts to
from the
cell to toxicity
can be
maintain
are often performed
the structural fractions,
conditions than can be attained
Although there have been several earlier in
reactive
conjugates with
elimination
compoundmetabolism and toxicity
experimental
from
content.
which is lost in tissue homogenates and subcellular for more controlled
cells
and forms S-substituted
compoundsto facilitate
glutathione
lines.
protect
provides reducing equivalents,
Thus, the susceptibility
Studies of xenobiotic in
It
(1).
and peroxides,
of xenobiotic
body or cell.
(GSH) can
reports of variability
changes in
been considered
GSH levels in
planning
integrity
while allowing in vivo (2,3). in GSHlevels due to
growth
or interpreting
studies.
Abbreviations used: FCS, fetal calf serum; GSH, glutathione; GSSG, glutathione and selenous acid Premix (Collaborative disulfide; ITS, insulin, transferrin, PBS, phosphate buffered saline. Research); NPSH, non-protein sulfhydryl; 0006-291X/83 737
$1.50
Copyright 0 1983 by Academic Press, Inc. At1 rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 114, No. 2, 1983
This report
AND BIOPHYSICAL
describes the effects
content in a humanlung (type II) examined were time after
RESEARCH COMMUNICATIONS
of varying
culture
tumor derived cell line,
passage, serum concentration,
combination of the growth factors,
insulin,
conditions
on GSH
A549. The parameters and the presence of a
transferrin,
and selenous acid.
and Methods
Materials
A549 (human type II lung tumor derived) cells were obtained from American Type Culture Collection (Rockville, MD). Cells were grown in Dulbecco's Modified Eagles Media (Gibco) with dialyzed fetal calf serum (FCS, Gibco) as indicated. Insulin (5 mg/l), transferrin (5 mg/l), and selenous acid (5 rig/l) were added as ITS premix (Collaborative Research) as indicated. Cells were used in early passages, determined to be mycoplasma free, and cultured in monolayer in the presence of kanamycin sulfate (100 W/ml, Gibco) in 5% CO2 in a humid atmosphere at 37O. Cells were passed by trypsinization when approaching confluence. For experiments in 2s or 10% FCS, cells were grown prior to passage in For media containing the same FCS concentration to be used in the study. studies in serum free media, cells were grown in 25 FCS and then replated at time 0 in media lacking FCS. For determination of cell number and GSH content, cells were washed with A hemocytometer was used for cell PBS and removed by trypsinization. counting. For GSH analysis, cells from one 75 cm2 flask were collected by washed with PBS and resuspended in 1 ml PBS. Protein was centrifugation, precipitated by addition of 50 ul 70% perchloric acid, followed by resuspension and centrifugation. GSH analysis was performed by the high pressure liquid chromatography method of Reed et al. (7). The method involves reaction of GSHwith iodoacetic acid (BDH Chemicals, Poole, England) to prevent GSH oxidation, followed by derivatization with 1-fluoro-2,4-dinitrobenzene (Aldrich) to form a colored product. Separations were performed on a 3-aminopropyl-Spherisorb column kindly supplied by Dr. D.J. Reed of Oregon State University. An Altex 420 gradient HPLC system equipped with a Spectra Physics SP4100 integrator was used. Sample injections were 100 ul, and effluent was monitored at 365 run. The detection limit for both GSHand GSSGusing this system was approximately 1 nmole. Student's t test was used to determine statistical significance. Results Table 1 shows both cell A549 cells point,
number and glutathione
grown in O%, 2%, or 70% FCS, with and without
GSH content
was higher
example, at 24 hours, GSHlevels 4.5
content per 106 cells
nmoles/lo6
cells,
24.2
as serum concentration for
cells
nmoles/lO6 cells,
and
In both 10% and 2% FCS, GSHlevels
hours after
passage and decreased at each time point 10%
At each time
was increased.
For
grown in O%, 2%, and 10% FCS were
respectively.
was more pronounced in
ITS.
for
120.8
nmoles/106
cells
increased between 0 and 24 thereafter.
This effect
than in 2% FCS. In 10%FCS, a 120%increase in GSH
occurred between 0 and 24 hours, while in 2% FCS, a 31% increased was observed. In serum free media, the
plating
efficiency 738
was reduced greatly,
from 758-808
Used:
-
-
8.5+ 0.4?
4.5t l.lb,c
15.8* 5.3
GSHa
-
0.34f 0.15
0.28t 0.10
0.91f 0.01
Cells (x106)
-g-
Time
Since
-
-
13.5* 5.3
5.9+ l.sb,c
15.8f 5.3
GSH
0% FCS+ITS
of
4.7i l.zd,e
6.54+ 0.76
passage
passage
15.3* 2.70
16.9t 3.?d
24.2+ 3.6e
18.5k 1.8d.e
GSH
A549
4oo;;
3;IO:; .
2.25f 0.09
$7;; .
Cells (x106)
in
Serum
Table
2% FCS
Levels
Passage,
aGSH expressed in nmoles/IOG cells bpcO.05 when compared to 0 hours in same media cpcO.05 when compared to 2% FCS at same time after dpcO.05 when compared to 24 hours in same media ept0.05 when compared to 10% FCS at same time after fN.A. = Not analyzed SExperiment terminated because cells lost attachment
96
~
0.322 0.07
48
-g-
0.15* 0.07
24
72
0.91* 0.01
Cells (x106)
0% FCS
0
Hours After Passage:
Media
Effects
I
7.8f 0.8d
12.7i 1.08
3.55+ 0.16
4.86* 0.68
15.6f 2.6
19.6k 4.3e
7.4f 2.ld
and
3.36+ 0.63
1.93t 0.35
3.3ot 0.14
GSH
2% FCS+ITS Cells (x106)
Cells
Concentration,
ITS
1.6Ot 0.55
l.Ol+ 0.15
N.A.f
0.54t 0.12
0.83i 0.02
Cells (x106)
37.2* 0.9d
51.6f 8.od
N.A.
120.8* 44.2
41.0f 5.8d
GSH
10% FCS
on GSH
31.4f 1.6d
6.8i o.gd
3.08t 0.94
N.A.
92.2* 12.0
42.Ok 1.d
GSH
1.93+ 0.74
N.A.
0.93f 0.28
1.13+ 0.22
Cells (x106)
10% FCS+ITS
Vol. 114, No. 2, 1983
in 2% or
BIOCHEMICAL
AND BIOPHYSICAL
10%FCS to 16% in serum free media.
in 0% FCS are equivalent 2% FCS prior
to passage.
plating
efficiency
the rate
Time 0 conditions for cells grown
to time 0 for 2% FCS, since cells
were maintained in
In serum free media, GSHcontent decreased between 0
and 24 hours, and then increased slightly ITS did not affect
RESEARCH COMMUNICATIONS
cell
growth,
between 24 and 48 hours. except
in the
case of 0% FCS, where
increased from 17% to 31% when ITS was present.
However,
of growth between 24 and 48 hours was the same in serum free media
with or without
ITS.
those grown without
GSHcontent was higher in cells only at 72 and $?6hours in
10%
grown with ITS than in
FCS.
However, the pattern
of time dependent decrease in GSHwas not altered by ITS. Since GSHlevels and decrease with availability variations 10%
appeared to increase with increasing
time after
serum concentration
passage, the question arose as to whether the
of GSH precursors in the media was responsible for the observed in GSH. To test
this idea,
cells
were plated in media containing
FCS, which was replaced with fresh media at 24 hour intervals.
illustrates
that
changing the media daily
Figure 1
did not prevent the time dependent
decrease in GSH. Additionally, by oxidation
the observed variations
in GSHlevel
cannot be accounted for
of GSHto GSSG. GSSGwas not detected in any cell
sample, nor was
any GSHor GSSGseen in the media in which the cells had been grown. o--o o- -0 M + 4
CELL NlJMIEU-CONTROL CELL NiJhf~¶Ek+- MEWA REPLACED GlUTArHlONECONWOL GUJTATHIONE - MEDIA RERACED
HOURS
OF GROWTH
Effects of changing media daily on growth and GSHcontent in A549 cells. Cells were grown in media containing 10%FCS, and media was removedand replaced with fresh media at 24, 48, and 72 hours. Values are mean2 S.D. for 3 flasks of cells. Fin..
740
BIOCHEMICAL
Vol. 114, No. 2, 1983
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I>iscussion The results varies
with
reported
time after
decreased thereafter observed that plating
CSH content
GSH increased
passage.
when cells
affects
GSH levels
were much lower in 2% than
it
in 2% and
was
Although
in A549 cells.
FCS, while in
10%
passage and
Additionally,
FCS.
of growth were similar
and rate
in A549 cells
24 hours after
were grown with
serum concentration
efficiency
levels
here demonstrate that
FCS, GSH
10%
free media, GSH
serum
content was reduced further. The combination of insulin,
and selenous acid sold as ITS
transferrin,
by
Collaborative
Research has been claimed to reduce the serum requirements for
many cultured
cells.
and
and that
10% serum,
concentrations.
Our results ITS
show that for A549, growth is similar
in 2%
does not
serum
Moreover, GSHcontent in
affect
Fluctuations glutathione
related
in
non-protein
In Ehrlich
9 hours after
hamster ovary cell cultures
with
ascites
transplantation NPSHlevels
fresh
media (6).
Thus ITS did not influence
(NPSH) content,
in maintained cell tumor cells,
within
of
lines
which
have been
NPSH levels
into a fresh host (4).
tripled
these
in GSH.
sulfhydryl
is the major component (8),
observed previously. within
variations
in
cells grown in 2% FCSwas much lower
than in 10%FCS, regardless of the presence of ITS. the serum concentration
growth
doubled
Similarly,
4 hours after
dilution
Chinese of cell
Passage of A549 cells
which are grown in
monolayer could be considered analagous to transplantation
to a fresh host or
dilution
with fresh media.
Harris and Teng (6) suggested that after
the rapid increases in cellular
passage described above were due to an increase in the proportion
proliferating phase cells.
cells, Our
which have a higher rate of protein results
decreases in GSH content
do not
with
reconcile
observed after
synthesis than plateau
this
theory,
media with
fresh
media containing 741
since
the
cells remains constant.
The time dependent decrease in GSHwas not prevented by daily culture
of
24 hours occurred during log-linear
growth, during which the percentage of proliferating
of
GSH
10%
replacement
FCS, the highest
serum
Vol. 114, No. 2, 1983
concentration free
BIOCHEMICAL
tested.
RESEARCH COMMUNICATIONS
The FCS used in these studies was dialyzed
amino acids and other
growth factors.
AND BIOPHYSICAL
low molecular weight compounds, such as peptide
The amino acid precursors
were present in excess in the media. in the media is unlikely
to remove
needed for
de novo GSH synthesis
Thus the availability
of GSHprecursors
to be responsible for variations
in GSH content with
time or serum concentration. Large differences
(greater
than 10 fold
occurred with time during log phase growth. it is important
at least for A549 cells,
in
10% FCS) in
GSH content
Thus, when reporting
GSHlevels,
to mention the time after
passage at
which measurements were made. Sumner and Wiebel (9) reported GSH levels 50.7 nmoles/mg protein
in A549 cells.
The cells
value of 8 nmoles/l06 cells
at 96 hours after
confluent,
to
In
conclusion,
conditions.
Serum
cellular
important
for
standardization xenobiotic
appears
interpreting of culture
is
markedly to
be
affected
highly
The mechanismof serum regulation
Since the metabolism and toxicity
mediated by the availability
passage, when cells were nearly
nmole/mg protein.
GSH content
concentration
determining GSHcontent. unknown.
69
were grown in 10% FCS and
This data corresponds well to our
were used when approaching confluence (10).
and is equivalent
of xenobiotic
studies of the effects
by culture
significant
of GSHis,
of GSH, knowledge of intracellular
in
however,
compounds can be GSHlevels is
of these compounds. Thus,
conditions is necessary when studying the effects
compoundson cultured
of
of
cells.
Acknowledaements: This work was supported by NIEHS Grant ES-02916 and by Cancer Center Grant 2-P30-CAI4236-llA1. References: 1. Meister. A. (1981) Biochem. Sot. Trans. 10, 78-79. (1977), in:eV.2. 2. J.R. and Bridges, J.W. Fry, J.W. Bridges and L.F. Chasseaud, ed., pp.71-118, Wiley, London. Tardiff, R.G. (197'8) Ann. Rev. Pharmacol. TOX~CO~.18, 357-369. Harris, J.W. and Patt, H.M. (1969) Exptl. Cell. Res. 56, 134-141. Ohara, H. and Terasima, T. (1969) Exptl. Cell. Res. 58, 182-185. Harris, J.W. and Teng, S.S. (1972) J. Cell. Physiol. 81, 91-96. Reed, D.J., Babson, J.R., Beatty, P.W., Brodie, A.B., Ellis, W.W., and 7. Potter, D.W. (1980) Anal. Biochem. 106, 55-62. 8. Tietze, F. (1969) Anal. Biochem. 27, 502-522. Sumnner, K.-H. and Wiebel, F.J. (1981) Toxicol. Letters 9, 409-413. 9. 10. Weibel, F.J. and Singh, J. (1980) Arch. Toxicol. 44, 85-97.
2 65:
142
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
July 29, 1983
Pages
737-742
EFFECTSOF CULTURE CONDITIONSONGLUTATHIONE CONTENT IN A549 CELLS Gloria B. Post, Douglas A. Keller,
Karen A. Connor, and D. B. Mensel
Departments of Pharmacology and Medicine, Comprehensive Cancer Center Duke University Medical Center Durham, North Carolina 27710 Received June
17,
1983
Summarv: The effects of varying culture conditions on glutathione content in A549 (human type II lung tumor derived) cells were examined. Parameters studied were growth time, serum concentration, and the presence or absence of a mixture of insulin, transferrin, and selenous acid. Glutathione content When cells were grown with serum, increased with serum concentration. glutathione increased sharply 24 hours after passage and decreased thereafter. Insulin, transferrin, and selenous acid had little effect on cell growth or glutathione content. Replacement of media with fresh media containing 10% serum did not prevent the growth dependent decrease in glutathione. These results demonstrate that glutathione content in A549 cells is strongly affected by culture conditions.
The tripeptide .
intermediates destroy
glutathione
in several ways
free radicals
metabolites
affected
by the cell's
cultured
cell
their
of a particular
Cultured
cells
maintained
conditions
cell
have not
metabolism and toxicity
lines
(4,5,6),
usually
acts to
from the
cell to toxicity
can be
maintain
are often performed
the structural fractions,
conditions than can be attained
Although there have been several earlier in
reactive
conjugates with
elimination
compoundmetabolism and toxicity
experimental
from
content.
which is lost in tissue homogenates and subcellular for more controlled
cells
and forms S-substituted
compoundsto facilitate
glutathione
lines.
protect
provides reducing equivalents,
Thus, the susceptibility
Studies of xenobiotic in
It
(1).
and peroxides,
of xenobiotic
body or cell.
(GSH) can
reports of variability
changes in
been considered
GSH levels in
planning
integrity
while allowing in vivo (2,3). in GSHlevels due to
growth
or interpreting
studies.
Abbreviations used: FCS, fetal calf serum; GSH, glutathione; GSSG, glutathione and selenous acid Premix (Collaborative disulfide; ITS, insulin, transferrin, PBS, phosphate buffered saline. Research); NPSH, non-protein sulfhydryl; 0006-291X/83 737
$1.50
Copyright 0 1983 by Academic Press, Inc. At1 rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 114, No. 2, 1983
This report
AND BIOPHYSICAL
describes the effects
content in a humanlung (type II) examined were time after
RESEARCH COMMUNICATIONS
of varying
culture
tumor derived cell line,
passage, serum concentration,
combination of the growth factors,
insulin,
conditions
on GSH
A549. The parameters and the presence of a
transferrin,
and selenous acid.
and Methods
Materials
A549 (human type II lung tumor derived) cells were obtained from American Type Culture Collection (Rockville, MD). Cells were grown in Dulbecco's Modified Eagles Media (Gibco) with dialyzed fetal calf serum (FCS, Gibco) as indicated. Insulin (5 mg/l), transferrin (5 mg/l), and selenous acid (5 rig/l) were added as ITS premix (Collaborative Research) as indicated. Cells were used in early passages, determined to be mycoplasma free, and cultured in monolayer in the presence of kanamycin sulfate (100 W/ml, Gibco) in 5% CO2 in a humid atmosphere at 37O. Cells were passed by trypsinization when approaching confluence. For experiments in 2s or 10% FCS, cells were grown prior to passage in For media containing the same FCS concentration to be used in the study. studies in serum free media, cells were grown in 25 FCS and then replated at time 0 in media lacking FCS. For determination of cell number and GSH content, cells were washed with A hemocytometer was used for cell PBS and removed by trypsinization. counting. For GSH analysis, cells from one 75 cm2 flask were collected by washed with PBS and resuspended in 1 ml PBS. Protein was centrifugation, precipitated by addition of 50 ul 70% perchloric acid, followed by resuspension and centrifugation. GSH analysis was performed by the high pressure liquid chromatography method of Reed et al. (7). The method involves reaction of GSHwith iodoacetic acid (BDH Chemicals, Poole, England) to prevent GSH oxidation, followed by derivatization with 1-fluoro-2,4-dinitrobenzene (Aldrich) to form a colored product. Separations were performed on a 3-aminopropyl-Spherisorb column kindly supplied by Dr. D.J. Reed of Oregon State University. An Altex 420 gradient HPLC system equipped with a Spectra Physics SP4100 integrator was used. Sample injections were 100 ul, and effluent was monitored at 365 run. The detection limit for both GSHand GSSGusing this system was approximately 1 nmole. Student's t test was used to determine statistical significance. Results Table 1 shows both cell A549 cells point,
number and glutathione
grown in O%, 2%, or 70% FCS, with and without
GSH content
was higher
example, at 24 hours, GSHlevels 4.5
content per 106 cells
nmoles/lo6
cells,
24.2
as serum concentration for
cells
nmoles/lO6 cells,
and
In both 10% and 2% FCS, GSHlevels
hours after
passage and decreased at each time point 10%
At each time
was increased.
For
grown in O%, 2%, and 10% FCS were
respectively.
was more pronounced in
ITS.
for
120.8
nmoles/106
cells
increased between 0 and 24 thereafter.
This effect
than in 2% FCS. In 10%FCS, a 120%increase in GSH
occurred between 0 and 24 hours, while in 2% FCS, a 31% increased was observed. In serum free media, the
plating
efficiency 738
was reduced greatly,
from 758-808
Used:
-
-
8.5+ 0.4?
4.5t l.lb,c
15.8* 5.3
GSHa
-
0.34f 0.15
0.28t 0.10
0.91f 0.01
Cells (x106)
-g-
Time
Since
-
-
13.5* 5.3
5.9+ l.sb,c
15.8f 5.3
GSH
0% FCS+ITS
of
4.7i l.zd,e
6.54+ 0.76
passage
passage
15.3* 2.70
16.9t 3.?d
24.2+ 3.6e
18.5k 1.8d.e
GSH
A549
4oo;;
3;IO:; .
2.25f 0.09
$7;; .
Cells (x106)
in
Serum
Table
2% FCS
Levels
Passage,
aGSH expressed in nmoles/IOG cells bpcO.05 when compared to 0 hours in same media cpcO.05 when compared to 2% FCS at same time after dpcO.05 when compared to 24 hours in same media ept0.05 when compared to 10% FCS at same time after fN.A. = Not analyzed SExperiment terminated because cells lost attachment
96
~
0.322 0.07
48
-g-
0.15* 0.07
24
72
0.91* 0.01
Cells (x106)
0% FCS
0
Hours After Passage:
Media
Effects
I
7.8f 0.8d
12.7i 1.08
3.55+ 0.16
4.86* 0.68
15.6f 2.6
19.6k 4.3e
7.4f 2.ld
and
3.36+ 0.63
1.93t 0.35
3.3ot 0.14
GSH
2% FCS+ITS Cells (x106)
Cells
Concentration,
ITS
1.6Ot 0.55
l.Ol+ 0.15
N.A.f
0.54t 0.12
0.83i 0.02
Cells (x106)
37.2* 0.9d
51.6f 8.od
N.A.
120.8* 44.2
41.0f 5.8d
GSH
10% FCS
on GSH
31.4f 1.6d
6.8i o.gd
3.08t 0.94
N.A.
92.2* 12.0
42.Ok 1.d
GSH
1.93+ 0.74
N.A.
0.93f 0.28
1.13+ 0.22
Cells (x106)
10% FCS+ITS
Vol. 114, No. 2, 1983
in 2% or
BIOCHEMICAL
AND BIOPHYSICAL
10%FCS to 16% in serum free media.
in 0% FCS are equivalent 2% FCS prior
to passage.
plating
efficiency
the rate
Time 0 conditions for cells grown
to time 0 for 2% FCS, since cells
were maintained in
In serum free media, GSHcontent decreased between 0
and 24 hours, and then increased slightly ITS did not affect
RESEARCH COMMUNICATIONS
cell
growth,
between 24 and 48 hours. except
in the
case of 0% FCS, where
increased from 17% to 31% when ITS was present.
However,
of growth between 24 and 48 hours was the same in serum free media
with or without
ITS.
those grown without
GSHcontent was higher in cells only at 72 and $?6hours in
10%
grown with ITS than in
FCS.
However, the pattern
of time dependent decrease in GSHwas not altered by ITS. Since GSHlevels and decrease with availability variations 10%
appeared to increase with increasing
time after
serum concentration
passage, the question arose as to whether the
of GSH precursors in the media was responsible for the observed in GSH. To test
this idea,
cells
were plated in media containing
FCS, which was replaced with fresh media at 24 hour intervals.
illustrates
that
changing the media daily
Figure 1
did not prevent the time dependent
decrease in GSH. Additionally, by oxidation
the observed variations
in GSHlevel
cannot be accounted for
of GSHto GSSG. GSSGwas not detected in any cell
sample, nor was
any GSHor GSSGseen in the media in which the cells had been grown. o--o o- -0 M + 4
CELL NlJMIEU-CONTROL CELL NiJhf~¶Ek+- MEWA REPLACED GlUTArHlONECONWOL GUJTATHIONE - MEDIA RERACED
HOURS
OF GROWTH
Effects of changing media daily on growth and GSHcontent in A549 cells. Cells were grown in media containing 10%FCS, and media was removedand replaced with fresh media at 24, 48, and 72 hours. Values are mean2 S.D. for 3 flasks of cells. Fin..
740
BIOCHEMICAL
Vol. 114, No. 2, 1983
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I>iscussion The results varies
with
reported
time after
decreased thereafter observed that plating
CSH content
GSH increased
passage.
when cells
affects
GSH levels
were much lower in 2% than
it
in 2% and
was
Although
in A549 cells.
FCS, while in
10%
passage and
Additionally,
FCS.
of growth were similar
and rate
in A549 cells
24 hours after
were grown with
serum concentration
efficiency
levels
here demonstrate that
FCS, GSH
10%
free media, GSH
serum
content was reduced further. The combination of insulin,
and selenous acid sold as ITS
transferrin,
by
Collaborative
Research has been claimed to reduce the serum requirements for
many cultured
cells.
and
and that
10% serum,
concentrations.
Our results ITS
show that for A549, growth is similar
in 2%
does not
serum
Moreover, GSHcontent in
affect
Fluctuations glutathione
related
in
non-protein
In Ehrlich
9 hours after
hamster ovary cell cultures
with
ascites
transplantation NPSHlevels
fresh
media (6).
Thus ITS did not influence
(NPSH) content,
in maintained cell tumor cells,
within
of
lines
which
have been
NPSH levels
into a fresh host (4).
tripled
these
in GSH.
sulfhydryl
is the major component (8),
observed previously. within
variations
in
cells grown in 2% FCSwas much lower
than in 10%FCS, regardless of the presence of ITS. the serum concentration
growth
doubled
Similarly,
4 hours after
dilution
Chinese of cell
Passage of A549 cells
which are grown in
monolayer could be considered analagous to transplantation
to a fresh host or
dilution
with fresh media.
Harris and Teng (6) suggested that after
the rapid increases in cellular
passage described above were due to an increase in the proportion
proliferating phase cells.
cells, Our
which have a higher rate of protein results
decreases in GSH content
do not
with
reconcile
observed after
synthesis than plateau
this
theory,
media with
fresh
media containing 741
since
the
cells remains constant.
The time dependent decrease in GSHwas not prevented by daily culture
of
24 hours occurred during log-linear
growth, during which the percentage of proliferating
of
GSH
10%
replacement
FCS, the highest
serum
Vol. 114, No. 2, 1983
concentration free
BIOCHEMICAL
tested.
RESEARCH COMMUNICATIONS
The FCS used in these studies was dialyzed
amino acids and other
growth factors.
AND BIOPHYSICAL
low molecular weight compounds, such as peptide
The amino acid precursors
were present in excess in the media. in the media is unlikely
to remove
needed for
de novo GSH synthesis
Thus the availability
of GSHprecursors
to be responsible for variations
in GSH content with
time or serum concentration. Large differences
(greater
than 10 fold
occurred with time during log phase growth. it is important
at least for A549 cells,
in
10% FCS) in
GSH content
Thus, when reporting
GSHlevels,
to mention the time after
passage at
which measurements were made. Sumner and Wiebel (9) reported GSH levels 50.7 nmoles/mg protein
in A549 cells.
The cells
value of 8 nmoles/l06 cells
at 96 hours after
confluent,
to
In
conclusion,
conditions.
Serum
cellular
important
for
standardization xenobiotic
appears
interpreting of culture
is
markedly to
be
affected
highly
The mechanismof serum regulation
Since the metabolism and toxicity
mediated by the availability
passage, when cells were nearly
nmole/mg protein.
GSH content
concentration
determining GSHcontent. unknown.
69
were grown in 10% FCS and
This data corresponds well to our
were used when approaching confluence (10).
and is equivalent
of xenobiotic
studies of the effects
by culture
significant
of GSHis,
of GSH, knowledge of intracellular
in
however,
compounds can be GSHlevels is
of these compounds. Thus,
conditions is necessary when studying the effects
compoundson cultured
of
of
cells.
Acknowledaements: This work was supported by NIEHS Grant ES-02916 and by Cancer Center Grant 2-P30-CAI4236-llA1. References: 1. Meister. A. (1981) Biochem. Sot. Trans. 10, 78-79. (1977), in:eV.2. 2. J.R. and Bridges, J.W. Fry, J.W. Bridges and L.F. Chasseaud, ed., pp.71-118, Wiley, London. Tardiff, R.G. (197'8) Ann. Rev. Pharmacol. TOX~CO~.18, 357-369. Harris, J.W. and Patt, H.M. (1969) Exptl. Cell. Res. 56, 134-141. Ohara, H. and Terasima, T. (1969) Exptl. Cell. Res. 58, 182-185. Harris, J.W. and Teng, S.S. (1972) J. Cell. Physiol. 81, 91-96. Reed, D.J., Babson, J.R., Beatty, P.W., Brodie, A.B., Ellis, W.W., and 7. Potter, D.W. (1980) Anal. Biochem. 106, 55-62. 8. Tietze, F. (1969) Anal. Biochem. 27, 502-522. Sumnner, K.-H. and Wiebel, F.J. (1981) Toxicol. Letters 9, 409-413. 9. 10. Weibel, F.J. and Singh, J. (1980) Arch. Toxicol. 44, 85-97.
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