104-109 - International Society for Fluoride Research Inc.

104
Fluoride Vol. 35 No. 2 104-109 2002 Research Report
EFFECTS OF FLUORIDE AND ASCORBIC ACID ON COLLAGEN
BIOSYNTHESIS IN MOUSE LIVER FIBROBLAST CULTURES
Maria Wardas,a Teresa Jurczak,b
Katarzyna Pawłowska-Góral,c Elżbieta Kotrys-Puchalskab
Sosnowiec, Poland
SUMMARY: Effects of fluoride on collagen biosynthesis were studied in
mouse liver fibroblast cultures in both the absence and presence of ascorbic
acid as an activator of lysyl hydroxylase and prolyl hydroxylase. The first series of cultures contained 0.12 mM NaF and a control without NaF. The second
series contained 0.12 mM NaF and 0.28 mM ascorbic acid, and the control
contained 0.28 mM ascorbic acid but no NaF. Collagen and non-collagen protein synthesis was estimated by incorporation of 14C proline, which indicated
a doubling of collagen biosynthesis and a 35-39% increase in non-collagen
protein in both series of experiments. The ratios of collagen with differing
solubility types in the cells and in the culture media were also altered, even
though the total amount of collagen in each series remained about the same
compared to the respective controls.
Keywords: Ascorbic acid, 14C proline, Collagen biosynthesis, Fibroblast cultures, Liver
fibroblasts, Mouse liver, Sodium fluoride.
INTRODUCTION
The biosynthesis and metabolism of collagen are affected by various
growth factors,1-4 collagen propeptides,5-7 arachidonic acid derivatives,8 several vitamins,9 and certain inorganic ions.10 Some of these promote or
stimulate collagen synthesis; others inhibit or interfere with it. Fluoride
compounds can behave in either way. As non-inert exogenous substances,
they can directly or indirectly disturb collagen biosynthesis or the action of
substances regulating collagen metabolism.
In this work we investigated effects of fluoride on collagen biosynthesis in
mouse liver fibroblast cultures in both the absence and the presence of
ascorbic acid as an activator of lysyl hydroxylase and prolyl hydroxylase.
MATERIALS AND METHODS
As described previously, livers of 60-day-old Balb c-strain mice of both
sexes were used.11ab Fibroblasts were isolated by tissue trypsinization and
cultured in Eagle’s medium MEM 1959 with the fetal calf serum containing
100 U of penicillin and 20 µg/mL of streptomycin in the atmosphere of 5%
CO2 at 310 K. All isolation and culture procedures were performed under
completely aseptic conditions in a laminar chamber and were conducted in
two sets of conditions. In series I the cultures contained 0.12 mM NaF with
a fluoride-free control. The series II cultured contained 0.12 mM NaF plus
———————————————
a
For Correspondence: Maria Wardas PhD, Department of Food and Nutrition, Silesian
Academy of Medicine, 4 Jagiellońska Street, 41-200 Sosnowiec, Poland; E-mail:
b
[email protected] The Institute of Experimental and Clinical Biochemistry,
c
Department of Chemistry and Biochemistry, Silesian Academy of Medicine; Department
of General and Analytical Chemistry, Silesian Academy of Medicine.
Fluoride and ascorbic acid effects on collagen biosynthesis
105
0.28 ascorbic acid and a control containing 0.28 mM ascorbic acid but no
fluoride.
Culture growth and protein content were controlled as we described previously.12 The intensity of collagen biosynthesis and its behaviour in cells
and culture media were monitored by measuring incorporation of 14Clabelled proline (2 µCi/mL). On the fourth day, 14C proline was added to the
experimental and control cultures, and after 48 hr the cultures were terminated. Essentially complete disruption of fibroblast suspensions was
achieved by sonication at 282 K for 1 min (10 sec bursts with 10 sec intervals to avoid heating). Centrifuging at 1400 g for 10 min at 282 K then gave
the supernatant for the different assays described below.
The amount of collagen biosynthesis in the fibroblast cultures was estimated by the method of Peterkofsky13 involving digesting the isolated proteins of collagenosis I. From this procedure a fraction of collagenosis digested protein (CDP) and a fraction of non-collagen protein (NCP) were
obtained. The relative collagen synthesis (RCS) was estimated by counting
the coefficient of its synthesis:
RSC=
CDP
⋅ 100%
(5.4 ⋅ NCP ) + CDP
The amounts of collagen formed were estimated by isolating and identifying three fractions from fibroblasts and the culture media by Grasedyck14
method: salt-soluble collagen (SSC), acid-soluble collagen (ASC), and insoluble collagen (ISC). The amount of collagen in each fraction was determined by measuring the 14C proline radioactivity, which was performed by
placing each fraction on a standard blotting disc and counting the beta decay
rate (dpm) with a Beckman scintillation counter. The counting efficiency
was 70-80%.
The one-way analysis test of variance was used to compare the mean values of all measurements. Differences with p<0.05 were considered significant.
RESULTS
As can be readily calculated from the data in Table 1, the amount of collagenosis digested protein (CDP) in both series I and series II was about
twice that in control cultures. On the other hand, the amount of non-collagen
protein (NCP) increased by only 43% in both series, and the coefficient of
collagen synthesis increased by 35% in series I and by 39% in series II.
Fluoride 35 (2) 2002
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Wardas, Jurczak, Pawłowska-Góral, Kotrys-Puchalska
Table 1. Content of 14C proline in collagen and
non-collagen proteins in mouse liver fibroblasts
Series
Type of
culture
-
I
II
*
Control 0 F
0.12 mM F
0.28 mM
ascorbic acid
0.12 mM F +
ascorbic acid
Collagenosis digested
protein (CDP ± SD)*
[dpm×10-3/mg prot.]
Non-collagen protein
(NCP ± SD)*
[dpm×10-3/mg prot.]
Relative collagen synthesis (RCS ± SD)*
[%]
20.08 ± 1.38
†
39.18 ± 1.78
160.13 ± 10.41
†
230.34 ± 17.60
2.27 ± 0.023
†
3.06 ± 0.093
19.97 ±1.61
161.11 ± 16.30
2.25 ± 0.061
†
†
40.09 ± 0.93
†
231.02 ± 15.97
3.13 ± 0.203
†
Values are average of 9 determinations. Compared with control group: p<0.05.
Table 2 shows the 14C proline levels of fibroblasts cell and culture media
fractions of differing solubility. In both series of experiments there was very
little difference in the net total collagen synthesis (cell plus media) between
the control groups of series I and series II as well as between the experimental groups of the two series. Nevertheless, in the controls of both series
the ratio of salt-soluble collagen (SSC) in the culture media to that in the
fibroblast cells was over 5:1, but in the experimental (fluoride-exposed)
groups this ratio was less then 4:1. Thus, in comparison to the controls, the
presence of 0.12 mM fluoride caused only an insignificant change in the total amount of growth (cells plus media) in the SSC fraction, and the same
was true of the acid-soluble collagen (ASC) fraction in series I.
Table 2. Content of 14C proline in individual collagen fractions
Series
I
II
*
Type of
culture
Salt-soluble collagen Acid-soluble collagen Insoluble collagen
(SSC± SD)*
(ASC± SD)*
(ISC± SD)*
[dpm×10-3/mg prot.] [dpm×10-3/mg prot.] [dpm×10-3/mg prot.]
Control
Medium
0 mM F
Cells
0.12 mM F Medium
Cells
133.03 ± 5.15
24.99 ± 0.52
†
143.91 ± 5.50
†
38.16 ± 2.36
10.28 ± 0.68
9.17 ±0.64
†
12.09 ± 0.66
†
9.89 ± 0.29
5.08 ± 0.40
3.19 ± 0.09
†
15.08 ± 1.01
†
4.98 ± 0.16
0.28 mM
Medium
ascorbic acid Cells
0.12 mM F + Medium
ascorbic acid Cells
135.25 ± 0.26
25.57 ± 0.32
†
143.00 ± 7.39
†
37.17 ± 2.87
13.79 ± 0.29
†
12.79 ± 0.09
†
12.49 ± 0.39
†
10.49 ± 0.19
†
5.10 ± 0.38
2.90 ± 0.10
†
16.80 ± 0.10
†
5.49 ± 0.27
†
Values are average of 9 determinations. Compared with control group: p<0.05.
Fluoride 35 (2) 2002
Fluoride and ascorbic acid effects on collagen biosynthesis
107
As also seen in Table 2, the amount of radiolabeled insoluble collagen
(ISC) in the culture media and the fibroblast cells was almost the same in the
controls in each series. On the other hand, the ISC in the culture media of
the two experimental (fluoride-exposed) series was about three times higher
than in the respective controls, and in the cells it was about twice as high as
in the controls.
From the data in Table 2 we can calculate the ratios of the values of SSC
to ISC fractions and ASC to ISC fraction as shown in Table 3. Note that the
SSC/ISC and the ASC/ISC ratios were slightly higher in the experimental
cell and media groups in series I than in series II. All these ratios, however,
were lower than those of respective controls in each series.
Table 3. Quantitative relations of salt soluble collagen
and acid soluble collagen to insoluble collagen
Series
I
II
*
Type of culture
SSC/ISC
ASC/ISC
Control
0 mM F
0.12 mM F
Medium
Cells
Medium
Cells
26.6
8.3
*
9.6
*
7.6
2.0
3.0
*
0.8
*
2.0
0.28 mM
ascorbic acid
0.12 mM F +
ascorbic acid
Medium
Cells
Medium
Cells
26.7
8.8
*
8.5
*
6.8
2.7
*
4.4
*
0.7
*
1.9
*
Compared with control group: p<0.05.
DISCUSSION
Comparison of the results with and without NaF permits unambiguous estimation of the influence of fluoride ions on collagen biosynthesis in the fibroblast cultures. Reciprocal quantitative relationships of individual collagen fractions between the cells and the media indicate that the amount of
14
C-labelled acid soluble collagen (ASC) as opposed to salt-soluble collagen
(SSC) and insoluble collagen (ISC) is not significantly changed when fluoride is present. In the presence of ascorbic acid (vitamin C) the amount of
ASC in the culture media is larger whether or not fluoride is present, although the increase is less with fluoride than without. It can be concluded
therefore that, in reference to the ASC fraction, ascorbic acid has some additional function apart from its known ability to stimulate the activity of lysyl hydroxylase and prolyl hydroxylase.
Fluoride 35 (2) 2002
108
Wardas, Jurczak, Pawłowska-Góral, Kotrys-Puchalska
Although the increase was relatively small, fluoride causes an increase in
collagen biosynthesis in the cells and the media of the SSC fraction. This
increase, however, was greater in the cells than in the media, even though
the SSC fraction in the media is much larger than in the cells. Fluoride therefore appears to promote the synthesis of proteins in the SSC fraction or to
inhibit their secretion into the media. In the light of Arka's work15 suggesting
that fluoride ions stimulate cellular extocytosis, it is reasonable to conclude
that the accumulation of SSC in fibroblasts as found here is result of increased biosynthesis of this fraction and not its impaired excretion.
In the two cultures with fluoride, the ISC cell fraction, although the smallest in quantity, showed the greatest relative amount of increase. In control
cultures the incorporation of labelled proline was greatest in the series I ISC
cell fraction without ascorbic acid.
An enhancement of ISC synthesis in these fibroblast cultures in the presence of fluoride is clearly demonstrated by the increase in radiolabelled proline incorporation into the ISC of both the media and the cells. This increase
modifies the post-translation collagen processes, and it has a leveling effect
on how much ascorbic acid can affect the activity of lysyl hydroxylase and
prolyl hydroxylase.
This work has unambiguously demonstrated that collagen biosynthesis is
enhanced by the fluoride and by about the same amount by ascorbic acid in
the presence of fluoride. What remains uncertain is how this occurs and how
fluoride may be affecting the biosynthesis or degradation of collagen.
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