Environmental Toxicology and Pharmacology 20 (2005) 501–505

Acetylsalicylic acid prevents nickel-induced collagen

biosynthesis in human fibroblasts
Wojciech Miltyk, Jerzy Palka ∗
Department of Medicinal Chemistry, Medical University in Bialystok,
Kilinskiego 1, 15-089 Bialystok, Poland

Received 4 January 2005; accepted 3 June 2005

Abstract

Exposure to nickel compounds that occurs mainly via inhalation can have adverse effects on human health. One of them is pulmonary
fibrosis that results from accumulation of collagen in lung tissues. The mechanism of this process as well as effective treatment of the disease
is not known. To evaluate the effect of nickel on collagen biosynthesis human dermal fibroblasts were treated with various concentrations of
nickel chloride(II) for 72 h. The compound was found to stimulate collagen biosynthesis in dose-dependent manner. We considered prolidase
as a potential target for nickel-dependent collagen biosynthesis regulation. Prolidase [E.C.3.4.13.9] is a cytosolic metalloproteinase, which
specifically splits imidodipeptides with C-terminal proline that is recycled for collagen biosynthesis. However, it was found that 72 h treatment
of confluent cells with Ni(II) did not affect significantly prolidase activity. An addition of acetylsalicylic acid, known, non-specific inhibitor
of prolidase to the cells treated with 100 ␮M NiCl(II), significantly reduced both collagen biosynthesis and prolidase activity. It suggests that
acetylsalicylic acid prevents nickel-induced increase in collagen biosynthesis through inhibition of prolidase activity in human fibroblasts.
The results indicate that tissue fibrosis may be considered as a possible target for prolidase inhibitory therapy and acetylsalicylic acid may
represent such an agent for potential application in tissue fibrosis prevention or early stages of tissue fibrosis.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Nickel; Prolidase; Collagen biosynthesis; Acetylsalicylic acid

1. Introduction One of the enzymes that play an important role in collagen

Nickel is widely used in modern industry. The high
consumption of nickel compounds leads to pollution of
environment. Human exposure to the nickel occurs mainly
via ingestion and inhalation. Nickel toxicity was found to
be associated with lung fibrosis (Berge, 2003), cardiovas-
cular and kidney diseases and cancer (Doll et al., 1970;
Grimsrud et al., 2003). Pulmonary fibrosis leads to progres-
sive lung destruction and scarring. The disease is character-
ized by increased number of inflammatory cells and excessive
tissue collagen deposition (Clark et al., 1982). The mecha-
nism of nickel-induced fibrosis as well as the effective treat-
ment of the disease is not known (Garantziotis et al., 2004).
∗ Corresponding author. Tel.: +48 85 7485702; fax: +48 85 7424907.
E-mail address: pal@amb.edu.pl (J. Palka).
biosynthesis is prolidase [E.C.3.4.13.9].
Prolidase is a metalloprotease requiring manganese for
catalytic activity. Prolidase catalyses the final step of colla-
gen degradation. The enzyme specifically splits imidodipep-
tides with C-terminal proline or hydroxyproline (Phang and
Scriver, 1989) and supplies free proline for collagen resyn-
thesis. Lack of the enzyme impedes the efficient recycling
of proline for collagen resynthesis (Jackson et al., 1975) and
cell growth (Emmerson and Phang, 1993). The efficiency of
recycling of proline for collagen biosynthesis was found to
be about 90% (Jackson et al., 1975). Previously, we found
the link between collagen synthesis and prolidase activity
in cultured skin fibroblasts treated with anti-inflammatory
drugs (Miltyk et al., 1996), during experimental aging of the
cells (Palka et al., 1996), fibroblasts chemotaxis (Palka et al.,
1997) and cell surface integrin receptor ligation (Palka and

1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.etap.2005.06.003
502 W. Miltyk, J. Palka / Environmental Toxicology and Pharmacology 20 (2005) 501–505
Phang, 1997). There is evidence that some anti-inflammatory
drugs evoke a decrease of collagen content in rat skin (Palka
and Galewska, 1990) and inhibits collagen biosynthesis in
cultured fibroblasts (Galewska et al., 1990). Therefore, the
present study was addressed to examine the effect of acetyl-
salicylic acid on collagen biosynthesis and prolidase activity
in cultured human dermal fibroblasts treated with well-known
inductor of tissue fibrosis, nickel chloride.
2. Materials and methods
2.1. Materials
Dulbecco’s minimal essential medium (DMEM) with
GlutaMax I, fetal bovine serum (FBS), penicillin and
streptomycin used in cell culture was products of
Gibco, USA. Prolidase, NiCl(II), collagenase (type VII
Clostridium Hystolyticum), dimethyl sulfoxide (DMSO),
methylthiazolyldiphenyl-tetrazolium bromide (MTT) and
acetylsalicylic acid (ASA) were obtained from Sigma, St.
Louis, USA. L-5[3 H] proline (28 Ci/mmol) was purchased
from Amersham, UK.
2.2. Tissue culture
All studies were performed on normal human dermal
fibroblasts, purchased in American Type Culture Collec-
tion (Manassas, Virginia, USA). Fibroblasts were maintained
in DMEM with GlutaMax I supplemented with 10% fetal
bovine serum, 50 U/ml penicillin, 50 ␮g/ml streptomycin at
37 ◦ C in a 5% CO2 incubator. Cells were counted in a hemo-
cytometer and cultured at 1 × 105 cells per well in 2 ml of
growth medium in 6 well plates (Sarsted, USA). Cells reached
confluence at day 4 and such cells were used for the assays.
Cells were used in the 8th to 12th passages. Fibroblasts were
incubated in medium with various concentrations of nickel
chloride(II) or nickel chloride(II) and acetylsalicylic acid for
72 h and used for experiments.
2.3. Cytotoxicity assay
Toxicity of Ni(II) was determined by method of Plumb et
al. (1989). Fibroblasts were maintained as described above.
After 72 h incubation of the cells with NiCl(II) the medium
was discarded and the cells were rinsed three times with phos-
phate buffered saline (PBS). Then the cells were incubated for
4 h in 2 ml of PBS with 50 ␮l of MTT (5 mg/ml). Medium was
removed from the wells, and the cells were lyzed in 200 ␮l
of DMSO with 20 ␮l of Sorensen’s buffer (0.1 M glycine
with 0.1 M NaCl, pH 10.5). The absorbance was measured at
570 nm.
2.4. Determination of prolidase activity
The activity of prolidase was determined according to the
method of Myara et al. (1982), which is based on measure-
ment of proline by Chinard (1952). Briefly, the monolayer
was washed three times with 0.15 M of NaCl. Cells were
harvested by scraping and suspended in 0.15 M NaCl, cen-
trifuged at low speed (200 × g) and the supernatant was
discarded. The cell pellet (from 2 wells) was suspended in
0.3 ml of 0.05 M Tris–HCl, pH 7.8 and sonicated three times
for 10 s at 0 ◦ C. Samples were then centrifuged (16,000 × g,
30 min) at 4 ◦ C. Supernatant was used for protein determina-
tion and then prolidase activity assay. Activation of prolidase
required incubation with manganese: 100 ␮l of supernatant
incubated with 100 ␮l of 0.05 M Tris–HCl, pH 7.8 contain-
ing 2 mM MnCl(II) for 2 h at 37 ◦ C. After pre-incubation, the
prolidase reaction was initiated by adding 100 ␮l of the incu-
bated mixture to 100 ␮l of 94 mM glycyl-proline (Gly-Pro)
for a final concentration of 47 mM Gly-Pro. After additional
incubation for 1 h at 37 ◦ C, the reaction was terminated with
1 ml of 0.45 M trichloroacetic acid. In parallel tubes reac-
tion was terminated at time “zero” (without incubation). The
released proline was determined by adding of 0.5 ml of the
trichloroacetic acid supernatant to 2 ml of a 1:1 mixture of
glacial acetic acid: Chinard’s reagent (25 g of ninhydrin dis-
solved at 70 ◦ C in 600 ml of glacial acetic acid and 400 ml
of 6 M orthophosphoric acid) and incubated for 10 min at
90 ◦ C. The amount of proline released was determined colori-
metrically by absorbance at 515 nm and calculated by using
proline standards. Protein concentration measured by the
method of Lowry et al. (1951). Enzyme activity was reported
as nanomoles per minute per milligram of supernatant
protein.
2.5. Collagen biosynthesis
The fibroblasts treated with Ni(II) or Ni(II) and acetylsali-
cylic acid were labeled for 24 h with 5[3 H]-proline (5 ␮Ci/ml,
28 Ci/mmol) and incorporation of radioactive precursor into
proteins was determined by digesting proteins with puri-
fied Clostridium histolyticum collagenase, according to the
method of Peterkofsky et al. (1982).
2.6. Statistical analysis
In all experiments, the mean values for six indepen-
dent experiments ± standard deviation were calculated. The
results were submitted to statistical analysis using Student’s
t-test, accepting P < 0.05 as significant.
3. Results
Confluent human skin fibroblasts were used to test the
effect of nickel chloride on collagen biosynthesis and pro-
lidase activity. The rational for using confluent cells in the
experiments was that collagen biosynthesis (Makela et al.,
1990) and prolidase activity (Myara et al., 1985) are depen-
dent on cell density and increase when the cell density
increases.
 W. Miltyk, J. Palka / Environmental Toxicology and Pharmacology 20 (2005) 501–505 503

Collagen biosynthesis was measured in fibroblasts treated

for 72 h with 50, 100 and 300 ␮M NiCl(II). The rational for
the prolonged treatment of fibroblasts with nickel is pre-
sented on Fig. 1C showing that maximal collagen biosyn-
thesis was found in the cells treated with 100 ␮M Ni(II)
for 72 h. As shown on Fig. 1A nickel chloride contributed
to the increase in collagen biosynthesis in dose-dependent
manner. At 50 ␮M NiCl(II) the process was increased by
50% and at 300 ␮M by over 75%. The effects were unre-
lated to the NiCl(II) toxicity. As shown on Fig. 1B, the
concentrations up to 300 ␮M NiCl(II) were not toxic for
confluent human fibroblasts. Increase of Ni(II) concentration
resulted in a reduction in the viability of the cells (data not
presented).

Fig. 2. Prolidase activity in confluent human fibroblasts treated for 72 h with

various concentrations of nickel chloride(II).

Since prolidase may play a critical role in collagen biosyn-

thesis the enzyme activity was tested in cell extract of fibrob-
lasts treated for 72 h with the same concentrations of NiCl(II)
as for collagen biosynthesis assay. Interestingly, there was no
correlation between increase in the collagen biosynthesis and
increase in prolidase activity. Nickel chloride at 50–300 ␮M
slightly inhibited the enzyme activity. At 300 ␮M the enzyme
activity was decreased only by about 10% (Fig. 2).
In order to test the effect of prolidase inhibitor on nickel-
induced collagen biosynthesis, we used acetylsalicylic acid,
known, non-specific inhibitor of prolidase activity (Miltyk

Fig. 1. (A) Collagen biosynthesis in confluent human fibroblasts treated for

72 h with various concentrations of nickel chloride (II). (B) The effect of
NiCl(II) on vialability of confluent human fibroblasts treated for 72 h with
various concentrations of NiCl(II). (C) Time course experiment for collagen
biosynthesis in fibroblasts treated with 100 ␮M NiCl(II). The mean values
of three independent experiment ± S.D. are presented.
Fig. 3. Prolidase activity and collagen biosynthesis in confluent human
fibroblasts treated for 72 h with 100 ␮M NiCl(II) and various concentrations
of acetylsalicylic acid (ASA).
504 W. Miltyk, J. Palka / Environmental Toxicology and Pharmacology 20 (2005) 501–505
et al., 1996). The cells were treated with 100 ␮M NiCl(II)
without and with various concentrations on ASA for 72 h.
Collagen biosynthesis in fibroblasts treated with 100 ␮M
NiCl(II) was increased by 50% compared to control cells
(Fig. 3). It has been found that 72 h treatment of confluent
fibroblasts with various concentrations of ASA contributed
to the decrease in prolidase activity and collagen biosynthesis
in a dose-dependent manner (Fig. 3). It was found that ASA
at 10 mM concentration prevented nickel-induced increase in
collagen biosynthesis by about 50%.
4. Discussion
The mechanism for nickel-induced fibrosis in humans is
poorly understood (Berge, 2003; Denkhaus and Salnikow,
2002). The data presented in this report suggest that non-
toxic concentrations of nickel chloride(II) stimulate colla-
gen biosynthesis in fibroblasts. We considered prolidase, as
a potential target for nickel-induced collagen biosynthesis
stimulation. However, the present study suggests that proli-
dase activity is not affected in nickel-treated fibroblasts.
The mechanism of nickel-dependent regulation of colla-
gen biosynthesis may take place at different levels. Exposure
of cells to nickel results in activation of HIF-1 transcrip-
tion factor and up-regulation of hypoxia-inducible genes
(Salnikov et al., 2002) including growth factors such as vas-
cular endothelial growth factor (VEGF) and platelet-derived
growth factor (Steinbrech et al., 2002; Namiki et al., 1995).
Both growth factors are responsible for collagen biosynthesis
induction (Amemiya et al., 1999; Pierce et al., 1991).
Treatment of cells with nickel may also produce hypore-
sponsiveness to IL-1, TNF-␣ and strong increase of NF-␬B
binding (Denkhaus and Salnikow, 2002). IL-1 and TNF-␣
are agents that are known to suppress collagen production.
IL-1 induces collagen degradation (Lu et al., 2004) while
TNF-␣ reduces collagen gene expression (Verrecchia and
Mauviel, 2004). The net result of both effects may contribute
to increase in collagen production. Another level of collagen
production may take place at the level of NF-␬B-induced col-
lagen biosynthesis (Lavrence et al., 2003). Increased binding
of the transcriptional factor induced by nickel compounds
may provide an explanation for mechanism of nickel-induced
fibrosis.
It cannot be excluded that the mechanism of nickel-
induced overproduction of collagen undergoes through estro-
gen receptor. Martin et al. (2003) demonstrated that nickel is
able to activate estrogen receptor alpha (ER-␣) and induce
expression of the estrogen-regulated genes in MCF-7 cells.
Our previous work showed a potent stimulatory effect of
estradiol on collagen biosynthesis (Miltyk et al., 1999). This
suggests that stimulatory effect of Ni(II) on collagen biosyn-
thesis may undergo through estrogen receptor.
Whether those various putative mechanisms contribute to
nickel-induced collagen accumulation in tissues remains to
be explored.
Although prolidase activity was not affected in NiCl(II)-
treaded fibroblasts, we decided to test the effect of prolidase
inhibitor on collagen biosynthesis in those cells. Khalil and
O’Connor (2004) used captopril, a potent prolidase inhibitor
(Ganapathy et al., 1985) as an anti-fibrotic agent in pul-
monary fibrosis. This data led us to test another inhibitor of
prolidase activity, acetylsalicylic acid (Miltyk et al., 1996).
As reported previously, ASA alone inhibit prolidase activ-
ity (Miltyk et al., 1996) and MMP (Karna and Palka, 2002)
and inhibit collagen biosynthesis (Miltyk et al., 1996). In
previous experiments we found that in fibroblasts NiCl(II)
at concentration 100 ␮M did not inhibit activity of MMP-2
and MMP-2 (data not published). Fibroblasts incubated with
100 ␮M nickel chloride elaborated about 50% more collagen
than control cells. Incubation of the cells with both nickel
chloride and ASA contributed to coordinate decrease in pro-
lidase activity and collagen biosynthesis. The decrease of
collagen biosynthesis was unrelated to the toxicity of the
compounds as detected by MTT test (data not presented).
The extend of the inhibition was dependent on ASA concen-
tration. It suggests that acetylsalicylic acid prevents nickel-
induced increase in collagen biosynthesis through inhibition
of prolidase activity in human fibroblasts. The results indi-
cate that tissue fibrosis may be considered as a possible
target for prolidase inhibitory therapy and acetylsalicylic
acid may represent such an agent for potential applica-
tion in tissue fibrosis prevention or early stages of tissue
fibrosis.
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