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Volume 45 Number 4 December 2012

The effect of nickel as a nickel chromium restoration corrosion 
product on gingival fibroblast through analysis of BCl-2

fX ady Soesetijo1 and Mandojo rukmo2
1Department of Prosthodontics, Faculty of Dentistry, Universitas Jember, Jember – Indonesia
2Department of Conservative Dentistry, Faculty of Dentistry, Universitas Airlangga, Surabaya – Indonesia

abstract

Background: Restoration of NiCr may undergo corrosion process in artificial saliva. Corrosion product is soluble Ni substances 
in salivary electrolytes. Ni2+ may freely enter the cells through passive transport DMT-1. Ni2+ in the cell causes initiation of the ROS 
formation,which subsequently can conduct the redoxs reactions leading to DNA damage. The damage DNA affects the genetic expression, 
especially bcl-2, and even triggers apoptosis. Purpose: The aim of this study was to reveal the mechanism of Ni toxicity as a corrosion 
product of NiCr restoration on gingival fibroblasts through expression analysis of Bcl-2. Methods: Cells with a density of 105 planted 
on each coverslip in 72 wells to the treatment group and 24 wells to the control group (24 hours incubation). In the treatment groups, 
each well exposed with 20 μL artificial saliva containing Ni concentration results immerse each restoration, whereas the control group 
was exposed to 20 μL artificial saliva (incubation 1, 3, and 7 days). The data collected were subsequently analyzed using two-ways 
ANOVA, followed by one-way ANOVA. Comparing between experimental groups after one-way ANOVA was conducted using Fisher’s 
LSD. Whereas, the calculation and documentation of Bcl-2 expression was performed camera of Olympus Microscope BX-50 Japan. 
results: Statistical analysis of two-ways ANOVA showed the presence of interaction between the increasing Ni concentration and 
exposure duration on the expression of Bcl-2 gingival fibroblasts (p=0.021<a=0.05). Conclusion: It can be concluded that the higher 
concentration of Ni exposed to gingival fibroblasts, and the longer incubation time will decreased Bcl-2 expression. 

Key words: Ni2+, DMT-1, DNA damage, Bcl-2

abstrak

latar belakang: Restorasi NiCr dapat mengalami proses korosi di dalam saliva artificial. Produk korosi yang dihasilkan 
adalah substansi Ni yang terlarut di dalam elektrolit saliva. Ni2+ bebas dapat memasuki sel (fibroblas gingiva) melalui transport 
pasif DMT-1. Ni2+ di dalam sel menginisiasi pembentukan ROS, yang selanjutnya dapat menjalankan reaksi redoks dan dapat 
menimbulkan kerusakan DNA. DNA yang rusak mempengaruhi ekspresi genetik, terutama Bcl-2 dan bahkan dapat memicu apoptosis.  
tujuan: Tujuan penelitian ini adalah untuk mengungkap mekanisme toksisitas Ni sebagai suatu produk korosi restorasi NiCr pada 
fibroblas gingiva melalui analisis ekspresi Bcl-2. Metode: Sel dengan kepadatan 105 ditanam pada tiap-tiap coverslip di dalam 72 
well untuk kelompok perlakuan dan ditanam pada tiap-tiap coverslip di dalam 24 well untuk kelompok kontrol (inkubasi selama 24 
jam). Pada kelompok perlakuan, masing-masing well dipapar dengan 20 μL saliva artificial yang mengandung konsentrasi Ni hasil 
perendaman tiap-tiap restorasi, sedangkan pada kelompok kontrol dipapar 20 μL saliva artificial (inkubasi 1,3 dan 7 hari). Data yang 
terkumpul selanjutnya dianalisis menggunakan ANOVA dua arah dan ANOVA satu arah. Perbandingan antar kelompok eksperimental 
setelah analisis ANOVA satu arah menggunakan uji Fisher’s LSD. Penghitungan jumlah sel yang mengekspresikan Bcl-2, kemudian 
dilanjutkan dengan dokumentasi dengan menggunakan kamera Olympus Microscope BX-50 Japan. hasil: Analisis statistik ANOVA dua 
arah menunjukkan adanya interaksi antara peningkatan konsentrasi Ni dan lama paparan terhadap ekspresi Bcl-2 fibroblas gingiva 

Research Report



203Soesetijo and Rukmo: The effect of nickel as a nickel chromium restoration corrosion product 

(p = 0,021 < á = 0,05). Kesimpulan: Dapat disimpulkan bahwa semakin tinggi konsentrasi paparan Ni pada fibroblas gingiva dan 
semakin lama masa inkubasi, maka akan menurunkan ekspresi Bcl-2. 

Kata kunci: Ni2+, DMT-1, DNA damage, Bcl-2

Correspondence: FX Ady Soesetijo, c/o: Departemen Prostodonsia, Fakultas Kedokteran Gigi Universitas Jember. Jl. Kalimantan 37 
Jember 68121, Indonesia. E-mail: adysoesetijo@ymail.com

introduction

Nickel-chromium (NiCr) alloy was widely used in 
dentistry for restoration construction purposes requiring 
strength, e.g. crowns, bridges and metal frame dentures. 
The consideration of the use of these alloys is mainly due 
to their relative low price compared to noble alloy, possess 
adequate hardness, and fine physical and mechanical 
properties. NiCr alloy consists of main components of Ni 
65%, Cr 22.5%, as well as additional components of Mo, 
Fe, Si, Ce and Nb.1-3

Biocompatibility NiCr restoration is a critical issue 
because of its existence in the long term of intimate contact 
with oral tissue. The presence of water, ions in saliva, 
changes in temperature and pH are factors that play a 
role in the occurrence of tarnish and corrosion. This was 
correlated with the release of alloy elements that impacts 
on unfavorable the biological effects.4 Corrosion may 
cause chemical changes of NiCr restoration. Chemical 
change occurring was an increase in restoration oxidation 
i.e. the formation of metal oxides easily detached from 
the surface of the restoration becoming cations. NiCr 
restoration recasting results in the increased toxicity in 
gingival fibroblasts due to the increase of Ni cations release 
in saliva.5,6

Ni is an unstable transition class and easy-released 
into saliva.7 NiCr alloy potentially causes toxic effects, for 
the reason that it may cause cellular damage. Ni elements 
released may affect the ability of cellular metabolism, 
particularly proliferation.8 Ni exposure on cultured 
cells may decrease the activity of several Fe-S enzymes 
associated with energy metabolism and induces oxidative 
stress by increasing the Reactive Oxygen Species (ROS). 
Generation of ROS in the cells affects the expression of 
B-cell lymphoma-2 (Bcl-2).9-11

Bcl-2 was a negative regulator of cell death, extends cell 
apoptosis and inhibits cell cycle. Bcl-2 family and the products 
were identified as a key regulator in the process of apoptosis 
in many cell types. Excessive expression of Bcl-2 increases 
cell survival. Apoptosis induced by Ni2+ is marked by the 
decrease of the expression of anti-apoptotic protein Bcl-2 and 
Bcl-xl, and induces the increase of the expression of Bad, Bcl-
xs, Bax, and caspase sit c (9,3 and 6).12 Ni2+ accumulation in 
cells can increase DNA damage response genes, decreased the 
expression of Bcl-2 gene and triggers apoptosis.13 Therefore, 
this study aims to determine the effect of toxicity of Ni as 
a product of NiCr restoration corrosion results in gingival 
fibroblasts through analysis of Bcl-2 expression.

materials and methods

This study began with a laboratory test to obtain a 
sample i.e the concentrations of Ni were dissolved in 5 
ml of artificial saliva after immersion of restoration NiCr 
casting, recasting 1× and 2× for 7 days.14 Restoration 
NiCr (diameter of 10 mm, thickness of 1 mm) obtained 
from casting alloys NiCr (4all NiCr alloy white ceramic, 
Ivoclar Vivadent-USA). Ni concentrations were obtained 
with the Atomic Absorption Spectrophotometer (AAS) 
assay (Model AG analytikjena ZEEnit 700), subsequently 
exposed on gingival fibroblast culture. Negative control 
group (describes artificial saliva containing no Ni on 
gingival fibroblasts). 

Gingival obtained from patients after conducting 
some procedures and filling out informed-consent, and 
subsequently undergoing gingivectomy. This study was 
approved by the ethical appropriateness of the research 
ethics committee of the Faculty of Medicine, Jember 
University Number: 125/H.25.I.II/KE/2011.

The next stage was to conduct the process of tissue 
culture. Cell cultures were grown in Dulbeco, s Eagle.s 
modified medium (DMEM) fetal bovine serum containing 
(FBS) 10% (v/v), penicillin-streptomycin 1% (v/v). The 
cells were then harvested from the culture dish/flask with 
0.025% trypsin-EDTA (Gibco). Cells with a density of 105 
planted on coverslip in 72 wells as treatment group and 
24 wells as control group (24-hour incubation), then the 
media were removed and washed with PBS. In the treatment 
group, each well was exposed to 20 mL artificial saliva 
containing Ni concentration resulted from the immersion 
of each restoration, whereas the control group was exposed 
to 20 mL artificial saliva (incubation 1, 3, and 7 days). 
Coverslip containing the cells were taken and placed on an 
object glass, then performed immunocytochemistry assay 
with Bcl-2 staining. 

Expression of Bcl-2 gingival fibroblasts was performed 
by immunocytochemistry assay with monoclonal antibody 
anti Bcl-2 (Bcl-2 kit: Bioworld, Cat. No. BS1511, USA). 
In this study, observations were carried out using light 
microscope (Olympus BX-50 Japan) with magnification of 
400 x, where the cells that expressed Bcl-2 in the cytoplasm 
was yellow-brown. Percentage of Bcl-2 = The number of 
positive cells Bcl-2 divided by the total number of cells 
x 100%.15 The data were analyzed by two way ANOVA, 
followed by one way ANOVA and multiple comparison 
Least Different Significance test with 95% significance 
level (a = 0.05).



204 Dent. J. (Maj. Ked. Gigi), Volume 45 Number 4 December 2012: 202–207

results 

Nickel solubility in artificial saliva performed by AAS 
with a wavelength of 323 nm, the parameters per parts 
million (ppm) and the units used are mg/L. The result is 
[Ni] casting = 0.780; recasting 1 x = 3.002 and recasting 2 
x = 6.320 (Tabel 1).

The mean and standard deviation of Bcl-2 expression 
progressively decreased with increasing Ni concentration and 
increasing duration of exposure. Based on this interpretation, 
it was necessary to analyze using two way ANOVA toward the 
possibility of interaction between increasing Ni concentration 
and duration of exposure (Table 2 and Figure 1).

The result of two way ANOVA showed that there were 
significant differences in the mean of Bcl-2 expression 
among the different concentrations of Ni (p < 0.0001), 
the mean expression of Bcl-2 also varied significantly 
among the durations of exposure (p < 0.0001), as well 
as the interaction between Ni concentration and duration 
of exposure (p = 0.021) (Table 3). The result of one way 
ANOVA showed that the expression of Bcl-2 gingival 
fibroblasts exposed to 0 mg/L (control), duration of exposure 
among the treatment groups are 1, 3 and 7 days, there 
was no difference between the mean of 1 day and 3 day-
exposures, while there were significant differences in the 
mean of 1 day and 7 days, and the mean of 3 days and 7 
days. Expression of Bcl-2 of gingival fibroblasts exposed 
to 0.780 mg/L shows that the mean of exposure duration of 
3 days and 7 days was not significant difference, whereas 
the mean and standard deviation of exposure duration of 
1 day and 3 days, as well as 1 day and 7 days demonstrate 
significant differences. In the mean time, expression of 
Bcl-2 gingival fibroblasts exposed to 3.002 mg/L shows that 
the mean exposure duration between 1 day and 3 days was 
not significant difference, whereas the mean of exposure 
duration between 1 day and 7 days, as well as 3 days and 
7 days shows significant differences. Expression of Bcl-2 

of gingival fibroblasts exposed to 6.320 mg/L shows that 
the mean of exposure duration between 1 day and 3 days 
was not varied, whereas the mean of exposure duration 
between 1 day and 7 days, as well as between 3 days and 
7 days demonstrates significant differences (Table 4 and 
Figure 2).

discussion

Rust was the result of corrosion, a form of metal oxides. 
Corrosion or rusting process was an electrochemical 
process. Restoration of NiCr acts as a reductant and O2 
dissolved in the saliva acts as an oxidant. Rust formed on 
restoration accelerates the subsequent corrosion process, 
therefore, rust was also known as autocatalysis. Oral 
environment was very conducive to the formation of tarnish 
and corrosion, as always wet and pH changes. Corrosion 
occurs when elements in the restoration of the lost electrons 
and ionized, so that the ions are formed is released into the 
saliva. Corrosion reactions in the oral cavity was aquaeus 
corrosion.

Genetic expression was an essential process activity 
in living organisms, as this process was the disclosure by 
the genes in the form of proteins, both structural proteins 
and protein enzymes. Structural support protein cellular 
components to perform its function, while protein enzymes 
catalyze change various compounds, including the genetic 
expression itself. Thus the process was the process of 

figure 1. The mean and standard deviation of Bcl-2 expression 
in the various [Ni] and duration of exposure.

table 1. [Ni] in the various restoration after immersion into 
artificial saliva for 7 days

Restoration [Ni] (mg/L)

Casting 0.780
Recasting 1x 3.002
Recasting 2 x 6.320

table 2. The mean and standard deviation of Bcl-2 expression 
in the various [Ni] and duration of exposure

[ni] (µg/l)

X ± Sd

duration of exposure (days)

1 3 7

0 18.75±1.49 17.63±1.41 15.38±1.69
0.780 10.25±1.04 8.00±1.07 7.50±0.93
3.002 4.88±1.13 4.25±0.46 3.25±0.71
6.320 2.88±0.83 2.50±0.76 1.75±0.89

table 3. The result of two way ANOVA of Bcl-2 expression 
in the various [Ni] and duration of exposure

Source df
Mean 
square

F P valued

Corrected model 11 298.030 252.874 <0.0001
Intercept 1 6272.667 5322.263 <0.0001
[Ni] 3 1060.250 899.606 <0.0001
Days 2 39.385 33.418 <0.0001
Interaction [Ni]*Days 6 3.135 2.660 0.021

r2 = 0.967

days



205Soesetijo and Rukmo: The effect of nickel as a nickel chromium restoration corrosion product 

isocitrate leading to decreased ATP production. Disruption 
of mitochondrial function is followed by a decrease or loss 
of expression of Bcl-2.

Transport Ni + + into cells may disrupt Fe + + homeostasis 
by inhibiting the extracellular iron into the cell, thereby 
inhibiting iron-dependent enzymes in the cytoplasm and 
mitochondria. Ni++ may also replace co-factor of Fe2+ in 
the prolyl hydroxylases, an enzyme playing a role in the 
activation of HIF-1a.16 Ni increases the activity of malate 
dehydrogenase (MDH) and isocitrate dehydrogenase (IDH). 
Increasing MDH and IDH reflect that there is an attempt 
to restore the function of the citric acid cell cycle. It was 
also explained that Ni exposure may increase the activity 
of glycolysis and the ratio of NADH/NAD. Cell oxidative 
phosphorylation depends on the iron to transfer electrons 
from NADH to oxygen, oxidative phosphorylation 
disruption result in the accumulation of NADH. Disruption 
of iron homeostasis results in ATP depletion and decreased 
expression of Bcl-2.11

Bcl-2 plays an important role in the regulation of 
programmed cell death apoptosis that was genetically 
regulated, where it was a balance between the expression 
of anti-apoptotic Bcl-2 and pro-apoptotic Bax. It showed 
a high degree of homology to Bcl-2. Over-expression of 
Bax leads to heterodimer bond with Bcl-2, as a result, 
Bcl-2 functions become inhibited and apoptosis induced. 
Whereas, if Bcl-2 was over-expressed, the protein will be 
bond with each other in homodimer, causing the cell to 
survive.17

The process of apoptosis is regulated through two 
pathways, extrinsic pathway (cytoplasm) through the 
activity of Fas death receptor by activating the Fas-
Fas ligand interactions (FasL), and intrinsic pathway 
(mitochondria), which triggers the release of sit-c which 
depends on the settings of Bcl-2 protein as an anti-apoptotic 
protein and Bax as pro-apoptosis protein.18

There are two key components that cause the release 
of sit-c, i.e. Mitochondrial permeability transition pore 
(MPTP) and apoptotic protein Bax. MPTP was located 
at the meeting point between the outer membrane and the 
inner mitochondrial membrane, which in an open state 
allowing compounds with a molecular weight less than 1.5 
KD pass freely between the matrix and the cytosol. The 
opening of MPTP is affected by Ca accumulation, oxidant 

table 4. The result of one way ANOVA of Bcl-2 expression in the various [Ni] and duration of exposure

[ni] (µg/l)
duration of exposure (days)

P valued
1 3 7

0 18.75±1.49a 17.63±1.41a 15.38±1.69b 0.001
0.780 10.25±1.04c 8.00±1.07d 7.50±0.93d <0.0001
3.002 4.88±1.13e 4.25±0.46e 3.25±0.71f 0.002
6.320 2.88±0.83g 2.50±0.76g 1.75±0.89h 0.038
P valued <0.0001 <0.0001 <0.0001

note: Vary of letter vary of significance (p valued<0.05) based on the result of multicomparison test

 

Figure 3. Bcl-2 expression (magnitude 400x) 
Notes : 
a. Exposure of 0,780 µg/L, incubated for 7 days, showed cells strong positive 

expression (         ) and negative expression (            ) 
b. Exposure of 3,002 µg/L, incubated for 7 days, showed cells weak positive expression 

(         ) and negative expression (            ) 
c. Exposure of 6,320 µg/L, incubated for 7 days, showed all of the cells negative 

expression  
d. Control,  incubated for 7 days, showed all of the cells strong positive expression  

 

figure 2. Bcl-2 expression (magnitude 400x).

Notes:
a. Exposure of 0.780 mg/L, incubated for 7 days, shower cells 

strong positive expression ( ) and negative expression 
( );

b. Exposure of 3.002 mg/L, incubated for 7 days, showed cells 
week positive expression ( ) and negative expression 
( ); 

c. Exposure of 6.320 mg/L, incubated for 7 days, showed all of 
the cell negative expression; 

d. Control, incubated for 7 days, showed all of the cell strong 
positive expression.

translating genetic expression of genes in the sequence of 
nitrogen bases form a protein amino acid sequence. Bcl2 
gene is one of the genes that contribute to carcinogenesis. 
The role of the expression of Bcl-2 inhibits apoptosis by 
ptotecting mitochondrial transition pore, so that the sit-c was 
not released. The role of Bcl-2 expression was in contrast 
to P 53, if P 53 is active, the Bcl-2 was inactive, whereas 
if P 53 is inactive Bcl-2 was active, so that apoptosis does 
not occur, and proliferation continues. This study suggests 
that there was the interaction between the Ni concentrations 
with the exposure duration on the expression of Bcl-2. 

The exposure to artificial saliva within 7 days may 
decrease the expression of Bcl2. It was caused by the 
anion present in the electrolyte, especially Cl- may affect 
Fe2+ transport into cells by forming FeCl2 compounds. It 
may cause disruption in the activities of Fe (II)-dependent 
enzyme that was important in aerobic catabolism in the 
mitochondria that was aconitase converting citrate into 



206 Dent. J. (Maj. Ked. Gigi), Volume 45 Number 4 December 2012: 202–207

and low mitochondrial transmembrane potential. MPTP 
is actually too small to be by passed by sit-c (13 Kd), but 
bonding with Bax forms a channel specifically for sit-c. 
The bonding between Bax and MPTP in pore formation 
activity is inhibited by anti apoptotic protein Bcl-2. So the 
balance among pro-apoptotic protein, anti-apoptosis and 
their interaction with MPTP largely determine the cells 
viability.19

Phagocytosis Ni by cells in the region of membrane 
ruffling, when there was a contact between Ni particles and 
the surface of cell membranes. The time required from the 
first contact up to the Ni endocytosis into cells is varied, 
it takes about 7–10 minutes. That process shows the effort 
salvatory motion of cells. Ni accumulation in and around 
the cell nucleus takes about 24–48 hours. Ni inside the 
cell may cause DNA fragmentation directly or indirectly 
through the formation of ROS. Damaged DNA stimulates 
the P53, which will further suppress the expression of Bcl-2 
via induction Bax.20

Ni compounds dissolved may affect intracellular Ca 
transport through a calcium ionophore ionomicyn channel, 
thus Ni was known as calcium channel blockers. Decrease 
in intracellular Ca concentration would lead to an increase 
in the compensation process from the free Ca supplies 
inside the cell (endoplasmic reticulum and mitochondria). 
Changes in the intracellular concentration signals the 
changes in the expression of Bcl-2.21,22 Ca possesses very 
important role, especially, its responsibility to regulate 
various physiological processes as well as involvement 
in maintaining homeostasis in pathological conditions 
and immune system. Therefore, it was also mentioned 
that the mitochondria serve as a buffer against Ca ions. 
Ca exits from endoplasmic reticulum along with sit-c 
release from mitochondria which was the phenomenon of 
apoptosis. Ca is identified as a message carrier to coordinate 
mitochondrial and endoplasmic reticulum that interact on 
apoptosis through suppression of Bcl-2, caspase activity, 
sit-c and nuclease enzyme.23, 24

Statistical analysis shows the determination coefficient 
of expression Bcl2 (r2 = 0.967). Thus, in this case the ability 
of the independent variables (Ni concentration and duration 
of exposure) in explaining the variance of the dependent 
variable (Bcl-2 expression gingival fibroblasts) amounted to 
97%, while the remaining 3% of variance of the dependent 
variable was caused by other factors.

Viewed from the size of the particles of each metal 
constructing NiCr alloys, then all of them may diffuse into 
the cell, because the size was smaller than the ion channels 
or pores of the membrane (300–400 Ao), which has a 
negative charge. When viewed from the electrode potential, 
the ability of the metal to retain or release electrons, the 
Ni2 + enters first because it possesses the highest electrode 
potential. The electrode potential possessed by Ni2+, Ni2 
+ causes a higher affinity for electrons or negative charge 
than the other cathions. Based on this, it may be presumed 
that the Ni2 + enters into cells via Ion channel divalent metal 
transporter-1 (DMT-1). The presence of Ni2 + in the cell may 

replace co-factor in the cytosolic enzyme, especially Mg 2 
+ co-factor required by G6PD. Eviction co-factor of Mg 2 
+ by Ni2 + causes the enzyme catalytic function was lost, so 
the anaerobic catabolism in the cytosol may not undergo, 
so that the next process in the mitochondria for aerobic 
catabolism may not undergo either, and make the ATP as 
a source of cellular energy may not be generated. 

The situation mentioned above may reduce the 
expression of Bcl-2. Another possibility was Ni2 + within 
cells act as free radicals that can cause oxidative stress 
in DNA. Injured/damaged DNA activates P 53 in the 
nucleus. P 53 stimulates and activates DNA repair genes. 
If the process of DNA repair does not succeed, then the 
P53 in the nucleus may be out into the cytosol. In the 
cytosol, P 53 will suppress Bcl-2 via activation of Bax, 
and eventually the cells will execute themselves through 
apoptosis mechanism.

Ni++ diffuse into the gingival fibroblasts presumably 
through DMT-1. In the cells, Ni++ directly or indirectly, 
through stimulation of ROS formation, cause injury/damage 
DNA. The injured/damaged DNA leads to the suppression 
of Bcl-2 with Bax activation, and eventually the cell 
executes themselves through the mechanism of apoptosis. 
It can conclude that the higher concentration of Ni exposed 
to gingival fibroblast, and the longer incubation time will 
decreased Bcl-2 expression.

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