CHEMISTRY
Ascorbic acid has a well documented role in collagen metabolism as a direct requirement for prolyl (1)and lysyl (2)
hydroxylases. It is required for hydroxylation of peptidylproline incell culture systelrns (3,4),unless a microsomal reducing
cofactor is present ( 5 ) .Ascorbic acid also stimulates collagen
production in cultured chick tendon (6, 7) and human skin
(8, 9) fibroblasts. Ascorbic acid increases procollagen mRNA
* This study was supported inpart by United States Public Health
Service Grants DK39996,DK07202, and DK38652 and by grants
from the Veterans Administration. The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Recipient of a Research Career Development Award, Veterans
Administration.
Supported in part by the Del Amo Foundation, Spain.
li Pew Scholar in the Biomedical Sciences.
16957
16958
Ascorbic Acid,
Lipid
Peroxidation,
and
culturing were obtained from the Core Cell Culture Facility (Univer- was eluted from a C18 column with 11%methanol (1 ml/min) after
sity of California, San Diego, CA).
5 min and measured at 546 nm.
Fibroblast Cultures-Human fetal AF, fibroblasts wereused at
Lipid peroxidation was also assessed in the cell layer by measuring
subcultivations 5-21 as previously described (14, 17). Cells were the 2,4-dinitrophenylhydrazinederivates of aldehydes formed form
cultured under an atmosphere of 5% CO,, 95% air in tissue culture the degradation of unsaturated fatty acids. Cells were harvested and
dishes using Eagle's minimal essential medium (MEM)' containing washed with phosphate saline buffer a t 0 "C, and reacted with 2,410% fetal calf serum. Cells were plated at a density of 9 X 106/P-10 dinitrophenylhydrazine. The derivates were separated by TLC as
dish, and radiolabeling studies were performed after 6 days as de- described by Benedetti and co-workers (35), but omitting the silicic
acid preparative chromatography. Polar and moderately polar fracscribed below.
Radiolabeling of Cells-Confluent cell cultures were incubated in tions were combined and analyzed separately from the nonpolar
10 ml of MEM with 10% fetalcalf serum containing 0.1 mM L-proline, carbonyl derivatives, by measuring their absorption at 365 nm. Values
a t 37 "C in 5% CO,; 95% air. The effects of lipid peroxidation on were expressed as nanomoles of carbonyls compounds produced using
collagen metabolism were examined by addition of ascorbic acid (0.2 the extinction coefficient of 25,500 (35).
Immunohistochemistry-Cells were grown to 60-80% confluency
mM) to plates at thetime of preincubation (0-72 h). In some experiments FeSO, (0.1 mM) was added to minimize variability in media on glass slides or in tissue culture slides (Lab-tek) and incubated for
fetal calf serum iron. Also, the influence of antioxidants, a-tocopherol 20 b in the presence of ascorbic acid (0.2mM) alone or with the
succinate (5-50 p ~ ) and
,
dimethyl sulfoxide (25 mM), as well as addition ofFeSO, (0.1 mM) and in the absence or presence of amethylene blue (10 p ~ )a, scavenger of reducing equivalents (18), on tocopherol (50 pM). Cells werefixed for less than 20 min in 4%
the ascorbic acid-treated cells was assessed by the addition of these paraformaldehyde, 20 p~ BHT, 2 mM EDTA, and 5% (w/v) sucrose.
compounds to the preincubation at the time of addition of ascorbic Guinea pig antibodies against specific malondialdehyde- and 4-hyacid. After the preincubation period, 0.2 mM ascorbic acid (a cofactor droxynonenal-lysine protein adducts were utilized as primary antifor prolyl and lysyl hydroxylases) and 740kBqof ~-[5-~H]prolinebodies to identify these modified proteins as evidence of in uiuo lipid
were added, and the incubation was continued for up to 4 hr (14). peroxidation (36). The primary antibodies were used in conjunction
The effects of exogenous malondialdehyde on collagen production with biotinylated goat anti-guinea pigIgG and the ABC-alkaline
was assessed by adding malondialdehyde (200 p ~ in) MEM without phosphatase systems essentially as recommended by the manufacfetal calf serum to thecell cultures at thetime of the incubation with turer. Nonspecific antibody reactions were assessed by omitting the
the radiolabeled proline. Labeling of cells was terminated by cooling first antibody.
Statistical Analysis-All the results are expressed as mean f S.E.
the plates to 0 "C.
Measurement of Collagen Production-Collagen and noncollagen unless stated otherwise. The Student's t test or analysis of variance
protein production in combined cell layer and medium were deter- were used to evaluate the differences of the means between groups,
acceptingp < 0.05 as significant (37).
mined by the collagenase method (19), as previously described (2022). The radioactivity of collagenase-sensitive and -insensitive proteins was used to calculate the relative rate of collagen production.
RESULTS
Eualuution of Cellular Tonicity-Loss of cellular lactic dehydrogenAscorbic Acid Induction of Lipid Peroxidation in Cultured
ase was measured in the supernatant of cell incubations. The lactic
dehydrogenase assay was carried out by a Cobas-Bio Analyzer. En- Cells-We assessed whether ascorbic acid induces lipid perzyme release was normalized from experiment to experiment by oxidation in cultured human fibroblasts. Incubation of cells
expressing the data as percent of total cellular lactic dehydrogenase with ascorbic acid induced the production of TBARS (Table
(23) as described previously (21). Intact cell counts were determined I), anindex of lipid peroxidation. This was determined by the
using a Coulter counter.
Preparation of cDNA Probes-The plasmid pHF677 (24) contain- thiobarbituric acid assay using either fluorometric detection
ing the cDNA for the human al(1) collagen was provided by Dr. F. or HPLC identification of the malondialdehyde-thiobarbituRamirez. The plasmid pKaI containing the cDNA for human a- ric acid complex. The addition of iron, but not ascorbic acid,
tubulin was provided by Dr. N. Cowan (25). The plasmid pcllase 1 at thetime of cell harvest led to a spurious increase in TBARS
containing the cDNA for human fibroblast collagenase was provided (data not shown). To assess lipid peroxidation in the presence
by Dr. P. Angel (26). The plasmid p k b 3 . u ~containing the human c- of additional iron in the preincubation period, the 2,4-dinitrofos cDNA (27) was provided by Dr. I. Verma. Using the random
phenylhydrazone derivatives of aldehydes formed from the
primer synthesis method (28), the cDNA fragments were radiolabeled
with [cP~*P]~CTPa specific
to
activity of approximately 1X lo9cpm/ degradation of unsaturatedfatty acids were measured. A
significant increase in nonpolar derivatives was found (6.4 f
pg of DNA.
Northern Blotting and Slot Blotting-AF, cells were incubated as 2 nmol/plate; p < 0.05) in cells preincubated with ascorbic
described above with ascorbic acid or malondialdehyde (200 p ~ )with
,
acid/FeS04. Similarly, there was an increase in polar and
or without a-tocopherol (5-50 p M ) or methylene blue (10 pM). Cells moderately polar carbonyl compounds. In addition, in cells
were processed as previously described (15), and RNA was isolated incubated with ascorbic acid we detected a marked increase
by the guanidine thiocyanate/phenol/chloroformextraction method
(29). Total RNA was usedfor Northern and slot blotting as
previously in malondialdehyde-protein adducts (Fig. 1B)and 4-hydroxdescribed (15, 30).The slot blot autoradiograms were quantitated by ynonenal-protein adducts (data not shown) by immunohisa scanning laser densitometer interfaced with an integrator. The level tochemistry utilizing specific antibodies against these aldeof collagen nl(1) mRNA in each sample was normalized to the level hyde-protein adducts. Sections incubated in the absence of
of a-tubulin mRNA.
the first antibody did not demonstrate any specific staining.
Run-off Transcription-The nuclear run-off transcription assay Therefore, both ascorbic acid alone and ascorbic acid/FeS04
which was developed by Groudine et al. (31) and adapted by Wang
and Calame (32) was used as previously described (15). The average
TABLE
I
experiment produced 1 cpm of radiolabeled RNA per nucleus.
Effects of ascorbic acid on lipid peroxidation
Determination of Lipid Peroxidation-Lipid peroxidation was determined by measuring thiobarbituric acid-reacting substances
Experimental condition"
TBARS
(TBARS) in the cell layer as described by Ohkawa and co-workers
nmol
(33). TBARS were measured fluorometrically (excitation 515 nm,
Control
0.29 & 0.04
emission 553 nm) using malondialdehyde and tetramethoxypropane
Ascorbic acid
1.01 f 0.08
standards. To some plates, FeS04 (0.1 mM) and/or ascorbic acid (0.2
Ascorbic acid/u-tocopherol
0.33 f 0.02
mM) wereadded before harvesting to assess their influence on TBARS
formation in the assay conditions. The thiobarbituric acid-malondiCells wereincubated for 20 h inMEM, 10% fetal calf serum alone
aldehyde complex was also determined by HPLC (34). This complex (control), or with the addition of ascorbic acid (0.2 mM) or ascorbic
acid, a-tocopherol (50 pM).
* Thiobarbituric acid-reactive substances were determined in cell
'The abbreviations used are: MEM, Eagle's minimal essential
medium; TBARS, thiobarbituric acid-reacting substances; HPLC, layers as described under "Experimental Procedures." Values are
means f S.E. of triplicate samples; p < 0.05.
high performance liquid chromatography.
16959
AA
CONTROL
Ascorbic Acid,
Lipid
Peroxidation,
and
16960
Collagen Gene
Expression
TABLE
I1
Noncollagen
proteinh
Collagenb
Experimental conditions"
dpm
96
Experiment 1
Control
3.0 f 0.1
95.0 f 4.1
100 f 5
Ascorbic acid/Fe*+
5.7 f 0.2
103.7 f 5.0
173 f 2
Fez+
3.5 f 0.2
107.0 f 4.4
103 f 2
Experiment 2
122.4 f 1.8
100 f 2
Control
9.3 f 0.3
109.5 f 9.8
151 f 11
Ascorbic acid
12.9 f 0.2
90.5 f 3.4
103 f 2
Ascorbic acid/a-tocopherol
7.1 f 0.3
Experiment 3
222.1 f 13.1
100 f 13
Control
5.2 f 0.9
269.3 k 26.0
196 f 17
Ascorbic acid
11.9 f 0.7
Ascorbic acid/methylene blue
3.1 f 0.3
183.0 f 20.4
74 f 4
a Confluent cell cultures were incubated for 20 h in MEM, 10% fetal calf serum, and labeled in the presence of
0.2 mM ascorbic acid for 4 h as described in Fig. 2. Experiment 1 , no additions (control)or with the addition of 0.2
mM ascorbic acid/FeSO, (0.1 mM) or 0.1 mM FeS04;Experiment 2, no additions (control) or with the addition of
;
3, no additions (control)or with the
ascorbic acid (0.2 mM) or ascorbic acid and a-tocopherol (50 p ~ )Experiment
addition of ascorbic acid (0.2 mM), or ascorbic acid and methylene blue (10 pM).
'Determined from the 'H radioactivity incorporated into collagenase-sensitive and -resistant proteins after
labeling cell cultures with 740 kBq of [5-'H]proline for 4 h. Values are means f S.E. of a t least triplicate samples.
p < 0.05 for collagen (ascorbic acid/Fe*+ (Experiment 1) and ascorbic acid (Experiments 2 and 3)).
Calculated from the formula ['H]collagen dpm/([3H]noncollagen dpm X 5.4 + ['H]collagen dpm) and expressed
as percentage of control values. The S.E. reported is the S.E. of the individual samples in each group.
Collagenb
Noncollagen
proteinb
&Iative
rate
of collagen
production'
dpm x
96
Control
9.7 f 0.6 529.0 f 33.2
100 f 6.0
178 f 16.0
Malondialdehyde 18.3 f 1.7 547.6 f 15.2
Confluent cell cultures were incubated in MEM, with or without
200 p~ malondialdehyde, and labeled in the presence of 0.2 mM
ascorbic acid and 740 kBq of [5-3H]proline for 3 h.
Determined as described in Table 11. Values are mean f S.E. of
triplicate samples; p < 0.05 for collagen.
Calculated as described in Table 11.
PROCOLLAGEN
5.5 kb
~i
Q1
FIG.3. Northern blot analysis of human fibroblast collagen mRNA. Confluent human fibroblasts were
incubated for 24 h in MEM, 10% fetal calf serum alone ( l a n e 1 ) with the addition of ascorbic acid (0.2 mM) ( l a n e
2 ) ; ascorbic acid and a-tocopherol (50 p ~ ( l)a n e 3 ) ;ascorbic acid and methylene blue (10 p M ) ( l a n e 4 ) ; ascorbic
acid (0.2 mM), FeS04 (0.1 mM) ( l a n e 5);or ascorbic acid, FeS04, anda-tocopherol (50 pM) ( l a n e 6).10 pg of total
RNA wereelectrophoresed on a formaldehyde 1%agarose gel and transferred to a nylon filter by capillary blotting.
The filters were hybridized to radiolabeled human collagen al(1)cDNA.
16961
16962
through another regulatory mechanism inhibits collagen gene 24. Chu, M.-L., Myers, J. C., Bernard, M. P., Ding, J.-F., and Ramirez, F.
(1982)Nucleic Acids Res. 10,5925-5933
expression (49,55).
25. Cowan, N. J., ,Dobner, P. R., Fuchs, E. V., and Cleveland, D.W. (1983)
Mol. Cell. Bwl. 3. 1738-1745
Lipid peroxidation is associated with the tissue injury and
P., Baumann, I., Stein, B., Delius, H., Rahmsdorf, H. J.,and
fibrogenesis of several pathological disorders, including ath- 26. Angel,
Herrlich, P. (1987)Mol. Cell. Bid. 7 , 2256-2266
27.
Mitchell,
R. L., Zokas, L., Schreiber, R. D., and Verma, I. M. (1985)Cell
erosclerosis (53), iron overload (56), porphyria (57), and
40.2119-21
,-.- - -7.
ethanol (58)-,bleomycin (59)-,and carbon tetrachloride (60)- 28. Feinberg, A. P., and Vogelstein, B. (1983)Anal. Biochem. 132,6-13
induced toxicity. The mechanisms by which tissue injury is 29. Chomczynski, P., and Sacchi, N. (1987)Anal. Bkchem. 162,156-159
30. Solis-Herruzo, J. A,, Brenner, D. A., and Chojkier, M. (1988)J. BioL Chem.
sometimes followedby fibrogenesis in uiuo are unknown.
263,584-5845
M., Peretz, M., and Weintraub,H. (1981)Mol. Cell. Biol. 3,281Whether they are related to the stimulation of collagen gene 31. Groudine,
9RR
expression induced by products of lipid peroxidation remains 32. Wa;, X.-F., and Calame, K. (1985)Cell 43,659-665
33. Ohkawa, H Ohishi N. and Yagi, K. (1979)Anal. Biochem. 96,351-358
to be established.
34.
Acknowledgments-We thank Michael Filip, Martina Buck, and
Linda Veloz for their excellent technical assistance and Kris Beaver
for her skillful preparation of this manuscript.
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