Mutat Res Gen Tox En 827 (2018) 19–26

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Mutat Res Gen Tox En

journal homepage: www.elsevier.com/locate/gentox

Corrosion potential in artificial saliva and possible genotoxic and cytotoxic T

damage in buccal epithelial cells of patients who underwent Ni-Cr based
porcelain-fused-to-metal fixed dental prostheses
⁎
Gülce Alpa, , Gonca Çakmakb, Murat Sertc, Yavuz Burgazd
a
Department of Prosthodontics, Faculty of Dentistry, Okan University, 34959, Akfırat, Istanbul, Turkey
b
Department of Toxicology, Faculty of Pharmacy, Gazi University, 06330, Hipodrom, Ankara, Turkey
c
Department of Medical Laboratory Techniques, Yildirim Beyazit University, 06760, Cubuk, Ankara, Turkey
d
Department of Prosthodontics, Faculty of Dentistry, Gazi University, Emek, 06510, Ankara, Turkey

A R T I C L E I N F O A B S T R A C T

The authors dedicate this article in the loving Nickel–chromium(Ni–Cr) based alloys account for the majority of the porcelain-fused-to-metal fixed dental
memory of Prof. Dr. Yavuz Burgaz, Professor of prostheses(PFM-FDPs) on account of their superior properties despite both nickel and chromium being known as
Department of Prosthodontics at Gazi human carcinogens. Understanding the genotoxicity and the cytotoxicity alongside the characteristics of cor-
University, who passed away on November 15, rosion behavior of the alloy is vital for understanding their biocompatibility. This study has evaluated whether
2017.
the Ni-Cr based alloys corroded in artificial saliva by analyzing alloy decomposition at different pH levels and
Keywords: immersion durations(7, 14, 21, and 28 days) using inductively coupled plasma-optic emission spectro-
Buccal epithelial cells photometry(ICP-OES). The principal aim of the study was to determine the possible genotoxicity and cyto-
Micronucleus
toxicity using micronucleus(MN) and other nuclear anomaly frequencies [nuclear bud(NBUD), binucleated
Corrosion
(BNC), condensed chromatin(CC), karyorrhectic(KhC), pyknotic(PC) and karyolytic(KC) cells] and various cy-
Salivary cotinine
Fixed dental prostheses tome parameters [basal cells(BC), differentiated cells(DF)] with the buccal epithelial cell(BEC) micronucleus
Ni-Cr alloys cytome assay(BMCyt). This test was administered at 1 pre- and 3 post-treatment time points to 40 patients who
underwent installation of PFM-FDPs made of Ni-Cr based alloy. Furthermore, at the final post-treatment time
point, saliva cotinine levels were measured with salivary cotinine quantitative enzyme immunoassay(EIA) kit
and information obtained by questionnaire prior to the first pre-treatment time point was confirmed. The highest
greatest release of Ni and Cr ions were seen at pH 2.3. MN and micronucleated cell frequencies, and BNC cell
frequencies were significantly elevated at post-treatment time points(p < 0.03). BC, CC, KhC, PC and KC cell
frequencies however were not significantly different between pre-and post-treatment time points(p > 0.05).
MN frequency was significantly lower in non-smokers than in current and former smokers(p < 0.001) at the
pre-treatment time point. There was no significant correlation between the unit number of PFM-FDPs and MN
frequencies. Our results revealed that Ni-Cr based alloys are prone to corrosion and that PFM-FDPs fabricated
with Ni-Cr based alloys may induce genotoxic effects rather than cytotoxic effect.

1. Introduction materials and techniques [5]. Consequently, good mechanical and

Porcelain-fused-to-metal fixed dental prostheses (PFM-FDPs), which
combine strength of metal and esthetic of porcelain, have been accepted
as a “gold standard” for many years and predominantly have been used
as an esthetic option in the restoration of teeth [1,2]. PFM-FDPs ex-
hibited good clinical performance characteristics with low failure rates
in follow up clinical studies over a minimum 5-year period [3,4]. The
success of the PFM-FDPs largely depends on the physical properties of
the metallic framework [5]. Metallic frameworks can be fabricated by
several types of dental casting alloys through improvements in
physical properties, casting techniques, cost, ease of workability, bio-
compatibility, and corrosion properties are the factors which affect the
selection of the alloy used [6,7].
Biocompatibility properties have an important role in the selection
of appropriate alloys for PFM-FDPs because these are placed in the
mouth and are in long-term contact with oral epithelial cells [8]. Un-
desired coloring, porosity and degrading of the mechanical properties
of the alloy occur as a result of corrosion [9]. The host response de-
pends on the type and the amount of the released elements as a result of
the process of degradation and the duration of exposure to these

⁎
Corresponding author.
E-mail addresses: gulce165@hotmail.com (G. Alp), msert06@gmail.com (M. Sert).

https://doi.org/10.1016/j.mrgentox.2018.01.004
Received 23 January 2017; Received in revised form 19 December 2017; Accepted 11 January 2018
Available online 12 January 2018
1383-5718/ © 2018 Elsevier B.V. All rights reserved.
G. Alp et al.
components [10]. Corrosion of alloys could be in constantly changing
amounts and affected by different factors [11,12]. In particular, the
mouth provides a conducive environment for the corrosion of metals
through occlusal load, pH and temperature alterations, bacterial ac-
tivity, dental plaque, diet, presence of moisture and saliva flow [13].
The corrosion of the alloy leads to the development of adverse biolo-
gical effects such as cytotoxicity, mutagenicity, carcinogenicity, and
allergic reactions [8,10]. Hence, apart from its mechanical and esthe-
tical properties, the corrosion resistance of an alloy is very important
for its biocompatibility [5,8].
Among the most used alloys, nickel-chromium (Ni-Cr) based alloys
are favored as the metallic framework material of PFM-FDPs due to
their low cost and good clinical performance [14]. Nickel and chro-
mium are metals capable of modulation of the immune response [15].
Moreover, both of the metals are known to either be human carcinogens
or possible carcinogens (IARC Group 1) [16,17]. Therefore, as they are
prone to corrosion, the safety of nickel-chromium (Ni-Cr) based alloys
in regard to the biocompatibility in the oral cavity is the subject of
toxicological evaluations, [11,12,18–21]. It has also been reported that
Ni-Cr based alloys have different corrosion potentials depending on the
type and pH of the corrosive medium, the immersion durations and the
chemical composition of the alloy [11]. Although there are in vitro
studies concerning the cytotoxicity and genotoxicity of this alloy
[16,22], to our knowledge, none have explored the cytogenic effects of
Ni-Cr based alloys used for PFM-FDPs construction.
Micronucleus assay as the measure of genotoxicity can be used in
several cell types from peripheral blood lymphocytes to the epithelial
cells such as buccal and nasal ones and in several areas from environ-
mental to clinical and to the occupational ones [23–26]. The technique
also allows for evaluating nuclear anomalies other than the micro-
nucleus frequency. As a result, it was named the cytome assay [27].
One of the aims of this study was to evaluate whether the Ni-Cr
based alloys were corroded in artificial saliva by analyzing decom-
position at different pH levels and immersion durations (7, 14, 21, and
28 days). A further aim was to determine the possible resultant geno-
toxicity and cytotoxicity by examining increases in the frequencies of
micronucleus (MN) and other nuclear anomalies [nuclear bud (NBUD),
binucleated (BNC), condensed chromatin (CC), karyorrhectic (KhC),
pyknotic (PC) and karyolytic (KC) cells] and cytome parameters [basal
cell (BC), differentiated cell (DF)] in buccal epithelial cell (BEC) mi-
cronucleus cytome assay (BMCyt) at 4 selected time points, pre- and
post- treatment, among a cohort of 40 patients who underwent proce-
dures to install PFM-FDPs made of Ni-Cr based alloy.
2. Materials and methods
2.1. Determination of elemental release in artificial saliva samples
A total of 36 Ni-Cr (Prestige KN, Adentatec Lagerei und
Warenvertriebs GmbH, Germany) rectangular samples with the di-
mensions of 32 mm × 15 mm × 1.2 mm were cast, grounded, and po-
lished according to the manufacturer recommendations. All samples
were prepared from new alloy without any surplus alloy. The samples
were immersed for 7, 14, 21 and 28 days at 37 °C in 3 different artificial
saliva samples with different pH values; sodium chloride and lactic acid
solution, pH 2.3 [10], Fusayama solution, pH 5.3 [28] and Ringer’s
solution, pH 7.0 [11]. The concentration of elemental release (Ni, Cr,
and molybdenum (Mo); ppm) was analyzed with static immersion test
method according to International Standards Organization (ISO)
10271:2001 [29] by using inductively coupled plasma-optic emission
spectrophotometry (ICP-OES) (Spectro Genesis, Kleve, Germany). All
measurements were repeated three times and the mean values were
calculated.
20
Mutat Res Gen Tox En 827 (2018) 19–26
2.2. Study design
2.2.1. Subjects
The subjects of this study comprised 40 healthy adult volunteers (20
men and 20 women) with a mean age of 34.2 ± 7.5 years, each of
whom had applied for prosthodontic treatment at the Department of
Prosthodontics, Gazi University, Turkey. Subjects completed a ques-
tionnaire scoring information on their age, sex, smoking status (current
smokers, former smokers, and non-smokers), alcohol consumption,
dietary habits, daily mobile phone usage time, X-ray exposure (over a
period of least one month previous to questionnaire completion), and
occupational and medical history. Current smokers were defined to be
those who had been smoking for at least 1 year before biological sam-
pling was undertaken. Former smokers were defined to be those who
had previously smoked cigarettes but had stopped smoking at least
1 year prior to sampling. None of the subjects had been exposed to
metallic alloys or other chemicals occupationally. Detailed clinical ex-
amination of the patients was performed. Subjects who had at least one
missing tooth and also had the indication for fixed dental prostheses
were included. However, subjects who had medical histories such as
genetic diseases, allergies, viral infections, a history of drug use, vac-
cination within the previous calendar year, oral lesions, prosthetic
treatment, amalgam restoration or periodontal disease, were excluded.
The Local Ethical Committee of Ankara University Faculty of Dentistry,
Ankara, Turkey (02.20.2012-30/7) approved this study. Prior to signing
a declaration of informed consent, each patient was thoroughly in-
formed about the purpose and the protocol of this study.
2.2.2. Characterization and application procedure of PFM-FDPs
The design and unit number of PFM-FDPs were defined according to
the number and the region of the missing tooth/teeth in the jaw, the
prognosis of the abutment tooth/teeth, the existence of parafunctional
habits and the esthetic expectations of the patient. A minimum of 3-unit
PFM-FDPs, made of Ni-Cr framework and feldspathic porcelain, were
each fabricated by the same technician, using a conventional protocol.
According to the treatment plan, the teeth were prepared so as to create
a chamfer finish line. Impressions for interim and definitive restorations
were made with irreversible hydrocolloid (Kromopan; Lascod SpA,
Firenze, Italy) and condensed silicone impression material (Zetaplus,
Zhermack SpA, Badia Polesine, Italy), respectively. Interim restorations
were fabricated on temporary casts and temporarily cemented (Cavex
Temporary Cement, Cavex, Haarlem, Netherlands) throughout the op-
erative procedures. In the laboratory, definitive casts were prepared,
mounted in a semi-adjustable articulator (Artex Type CT Articulator,
Jensen Dental, North Haven, USA) and Ni-Cr (Prestige KN, Adentatec
Lagerei und Warenvertriebs GmbH, Germany) metallic frameworks
were fabricated from new alloy. The marginal fit of the frameworks was
intra-orally evaluated and then sent to the laboratory for the fabrication
of the porcelain veneer. Feldspathic porcelain (Ceramco® 3, Dentsply,
New York, USA), compatible with Ni-Cr alloy, was applied on the fra-
mework and occlusal adjustments were performed intraorally. The re-
storations were permanently cemented with glass ionomer cement
(Ketac-Cem; 3 M ESPE, Seefeld, Germany) after glazing. The composi-
tion of the materials used in this study was presented in Table 1.
2.3. Biological sampling
To evaluate the possible genotoxic and cytotoxic effects of PFM-
FDPs, the BMCyt assay was used in pre- and post-treatment evaluation
of the subjects. BEC samples were collected in 4 different sampling time
points. The pre-treatment samples were collected immediately prior to
the operative procedure (1st sampling). In the post-treatment (after
cementation of the Ni-Cr based PFM-FDPs), the samples were collected
a further 3 times; 1 week (2nd sampling), 1 month (3rd sampling), and
3 months (4th sampling) after the cementation of the PFM-FDPs. Each
sampling simultaneously harvested a minimum volume of 3 ml
G. Alp et al. Mutat Res Gen Tox En 827 (2018) 19–26

Table 1
Composition of materials.

Material Manufacturer Composition%

Prestige KN Adentatec Lagerei und Warenvertriebs GmbH, Ni, 62.0; Cr, 25.0; Mo, 10.9; Si, 1.5; Fe, > 0.1
Koeln, Germany
Ceramco® 3 Dentsply, New York, USA Feldspathic porcelain with organic pigments, Sodium Potassium Aluminosilicate, Tin Oxide
Temdent Classic Schütz Dental GmbH, Rosbach, Germany Poly methyl methacrylate, copolymer methyl methacrylate
Cavex Temporary Cement Cavex Holland BV, Haarlem, Netherlands The base: A mixture of a vegetable oil, magnesium oxide and zinc oxide; The catalyst: a dimer fatty
acid in a mixture of waxes and resins as paste formers, a trace of acid
Ketac-Cem 3 M™ ESPE™, Seefeld, Germany Powder: Glass powder, polycarboxylic acid pigments; Liquid: Water, tartaric acid conservation
agents

Table 2
General characteristics of the subjects.

Men Women Total

(n = 20) (n = 20) (n = 20)

Age (Mean ± SD) 35.4 ± 8.4 33.1 ± 6.4 34.2 ± 7.5

Smoking habits n (%) Non-smokers 9 (45%) 10 (50%) 19 (47.5%)
Current smokers 6 (30%) 6 (30%) 12 (30%)
Former smokers 5 (25%) 4 (20%) 9 (22.5%)
Saliva cotinine (ng/ml) 84.8 ± 167.7 108.8 ± 266.0 96.8 ± 219.8
Daily cell phone usage time (Min) 32.0 ± 27.5 46.0 ± 63.2 39.0 ± 48.6
Dietary habits n (%) Vegetablesa 6 (30%) 14 (70%) 20 (50%)
Meat 14 (70%) 6 (30%) 20 (50%)
X-ray n (%) (periapical/panoramic) Yes 12 (60%) 11 (55%) 23 (57.5%)
No 8 (40%) 9 (45%) 17 (42.5%)
PFM-FDPs unit number (Mean ± SD;Min-Max) 6.4 ± 3.8; 3–15 5.3 ± 2.5; 3–11 5.8 ± 3.2; 3–15

SD, standard deviation; Min, minimum; Max, maximum; PFM-FDPs, porcelain-fused-to-metal fixed dental prostheses.
a
p = 0.027 significantly different (men vs women); Mann-Whitney U Test, Chi Square test.

unstimulated saliva specimens [30] from each subject to measure pH
levels immediately. At the 4th sampling time point, the saliva samples
were stored at −80 °C until the measurement of saliva cotinine levels in
order to verify smoking status [31]. All the biological samples were
taken before administration of local anesthesia. If periapical/panoramic
radiography (X-ray exposure) was needed before the 1st sampling, the
sampling was carried out at least 21 days after the X-ray exposure, so as
to give sufficient time for BEC turnover [32] to occur, and those who
were exposed to X-rays are indicated in Table 2. None of the patients
were exposed to intra/extra oral X-ray after the 1st sampling.
After obtaining the saliva samples, BEC were collected with a pre-
moistened wooden tongue-depressor by gently scraping both sides of
the right/left buccal mucosa. This material was then smeared over two
separate pre-cleaned, blindly coded, and pre-moistened microscope
slides. The smears were fixed in 80% methanol for 10 min, air dried,
and stored at room temperature until staining for BMCyt.
2.3.1. pH measurement in saliva samples
The saliva pH was measured using pH indicator papers immediately
after saliva was sampled (Universal Indicator, pH 0–14, Merck KGaA,
Darmstadt, Germany) [33].
2.3.2. Saliva cotinine analysis
Saliva specimens from the 4th sampling period were thawed and
centrifuged at 1500g (3000 rpm) for 15 min to remove debris and pre-
vent bacterial colonization. The salivary cotinine quantitative enzyme
immunoassay (EIA) kit (Salimetrics, State College, USA) and a micro-
plate reader were used according to the manufacturer’s instructions to
measure cotinine concentrations (ng/ml) present in saliva specimens
[30].
2.3.3. Buccal micronucleus cytome assay (BMCyt)
Slides were stained with Feulgen Reagent and counterstained with
Fast Green. The staining, identification and scoring procedure used in
this assay was done according to Thomas et al. [34]. The numbers of
basal (BC), differentiated (DF), binucleated (BNC), condensed chro-
matin (CC), karyorrhectic (KhC), pyknotic (PC) and karyolytic (KC)
cells were scored per 1000 cells. The mean frequency of MN (MN fre-
quency), the mean frequency of cells bearing at least one micronucleus
(Micronucleated cell frequency), and nuclear bud (NBUD) frequency
were scored in at least 2000 differentiated cells by the same scorer
blindly. Evaluations were performed by light microscopy at 400 x
magnification and confirmed at 1000 x magnification (Zeiss Axioscope
2 Microscope, Goettingen, Germany). The frequencies of each type of
cellular aberration observed were expressed per thousand [34].
2.4. Statistical analyses
All statistical analyses were performed using SPSS 20.0 (IBM SPSS
Statics, New York, USA). Data were shown as mean ± standard de-
viation (SD) for continuous variables, when appropriate. Nominal data
were evaluated using Fisher’s exact test. Wilcoxon Sign Test was used
for the differences among the time points while evaluating the variables
not normally distributed. Degrees of associations between variables
were analyzed using the Chi Square test. Where applicable with regard
to the number of independent groups, comparisons of median values,
the Mann–Whitney U test or the Bonferroni corrected Kruskal Wallis H
test were applied. Multiple linear regression analyses with adjustment
for potential confounding factors such as sex, age, smoking status
(smoker/nonsmoker), X-ray exposure (yes/no), dietary habits (con-
sumption of vegetables/meat), pH and mobile phone usage time were
performed to determine the effect of individual factors on MN fre-
quencies after log transformation for parameters which were not nor-
mally distributed parameters. Coefficients of regression and 95% con-
fidence intervals for all independent variables were calculated. The
correlations between MN frequencies and saliva pH, PFM-FDPs unit
number, and the cotinine concentration in saliva cotinine were per-
formed by Pearson correlation analysis. All statistical analyses were
performed using a significance level of p < 0.05.

21
G. Alp et al. Mutat Res Gen Tox En 827 (2018) 19–26

Table 3 Table 4
In vitro elemental release levels at different pH levels and after different immersion BEC genotoxicity parameters, the cytokinetic defects, proliferative potential and cell
durations. death (mean ± SD) in each pre- and post- treatment sampling time points of the study
subjects due to PFM-FDPs.
Immersion Ni (ppm) Cr (ppm) Mo (ppm)
durations (days) (n = 12) (n = 12) (n = 12) Frequency (‰) Pre-treatment Post-treatment

pH 2.3 7 4.0 ± 1.9a,b 0.9 ± 0.5b 0.3 ± 0.2a,b 1st sampling 2nd sampling 3rd sampling 4th sampling
14 8.4 ± 3.6a,b 2.6 ± 1.8a 1.8 ± 0.9
a
21 14.1 ± 3.2a 3.6 ± 0.8a 2.1 ± 0.3 MN frequency 4.0 ± 1.5 7.3 ± 1.9 7.5 ± 1.8 7.5 ± 1.8
28 16.9 ± 4.5a 4.3 ± 1.2a 2.9 ± 0.2b Micronucleated 3.6 ± 1.5a 6.2 ± 1.9b 6.7 ± 2.1 6.4 ± 2.0
pH 5.3 7 1.1 ± 0.6b 0.0 ± 0.0a,b 0.0 ± 0.0a,b cell frequency
14 3.5 ± 2.0 0.4 ± 0.7 1.7 ± 0.5b NBUD 1.5 ± 0.8c 1.5 ± 0.8 1.7 ± 0.8 1.8 ± 0.9
21 4.1 ± 2.4 0.5 ± 0.8 2.7 ± 0.3a BC 14.1 ± 3.0 14.6 ± 3.1 14.6 ± 2.8 14.9 ± 3.0
28 3.7 ± 1.5 0.4 ± 0.2 2.9 ± 0.4 BNC 11.4 ± 3.2a 13.6 ± 3.3 13.5 ± 2.9 13.4 ± 3.6
pH 7.0 7 2.1 ± 1.8 0.5 ± 0.3 0.0 ± 0.0b CC 47.2 ± 13.1 48.2 ± 10.8 47.7 ± 10.8 48.4 ± 14.2
14 2.3 ± 2.9 0.4 ± 0.3 0.5 ± 0.2a,b KhC 29.3 ± 9.1 29.4 ± 8.2 29.7 ± 8.6 30.2 ± 9.7
21 3.5 ± 4.0 0.1 ± 0.2a,b 2.2 ± 0.4 PC 14.2 ± 4.2 13.9 ± 3.5 13.9 ± 3.6 13.9 ± 3.0
28 3.3 ± 4.5 0.9 ± 1.4 2.1 ± 0.4a KC 64.1 ± 20.0 65.9 ± 16.2 68.2 ± 18.3 71.2 ± 21.4

SD, standard deviation; significantly different.
a
p < 0.001, element release at the pH of the artificial saliva vs element release at the
other two pH levels of the artificial saliva for the same immersion duration.
b a
p < 0.05, immersion duration vs all other immersion durations for the same pH,
Kruskal Wallis H Test.
3. Results
3.1. Elemental release levels in artificial saliva samples
In vitro elemental release levels in each of the three different pH
values and after 7, 14, 21, 28 days immersion were shown in Table 3.
Ni release in the artificial saliva was significantly higher at pH 2.3
than at pH 5.3 and pH 7.0 for all immersion durations (p < 0.05,
Table 3). Cr release was significantly higher at pH 2.3 than at pH 5.3
and pH 7.0 after 14, 21, and 28 days of immersion (p < 0.05, Table 3).
Mo release was significantly higher at pH 2.3 than at pH 5.3 and pH 7.0
after 7 days of immersion (p < 0.05, Table 3). Cr release was sig-
nificantly lower at pH 5.3 than at either pH 2.3 or pH 7.0 after 7 days of
immersion (p < 0.05, Table 3). Mo release was significantly higher at
pH 5.3 than at either pH 2.3 or pH 7 after 21 days immersion
(p < 0.05, Table 3). Additionally, Mo release was significantly lower at
pH 7 than at either pH 2.3 or pH 5.3 after 14 and 28 days of immersion
(p < 0.05, Table 3).
The release of Ni, Cr and Mo were found to be significantly lower
after 7 days of immersion than after 14, 21, or 28 days in artificial saliva
of pH 2.3 or pH 5.3 (p < 0.05, Table 3). Additionally Mo release was
found to be significantly lower after 7 days of immersion than after 14,
21, or 28 days in artificial saliva of pH 7 (p < 0.05, Table 3). Ni release
was significantly lower after 14 days of immersion than after either 21
or 28 days at pH 2.3 (p < 0.05, Table 3). Mo release was also sig-
nificantly lower after 14 days of immersion than after 21 or 28 days at
pH 5.3 or pH 7 (p < 0.05, Table 3). Mo release, however, was sig-
nificantly higher after 28 days of immersion than after either 14 or
21 days at pH 2.3 (p < 0.05, Table 3).
3.2. Demographic characteristics
The demographic characteristics of the subjects were shown in
Table 2. Age, smoking habits, saliva cotinine, daily cell phone usage
time, X-ray exposure (periapical/panoramic) values were found not to
vary in a significantly manner between men and women (p > 0.05).
Dietary habits were found to be a significant difference between men
and women (p < 0.05). The unit numbers of PFM-FDP were also si-
milar between men and women and the range was from 3 to 15
(p > 0.05). In the present study, there were 19 nonsmokers, 12 current
smokers and 9 former smokers. None of the subjects had consumed
alcohol, nor presented with a history of allergy and/or underwent
vaccination within the previous year.
SD, Standard deviation; MN, micronucleus; NBUD, nuclear buds; BC, Basal cell; BNC,
Binucleated cell; CC, Condensed chromatin cell; KhC, Karyorrhectic cell; PC, Pyknotic
cell; KC, Karyolytic cell.
p < 0.001, 1st sampling vs post-treatment time points.
b
p < 0.05, 2nd vs 3th sampling.
c
p < 0.05 1st vs 4th sampling.
3.3. pH levels of saliva samples
There were no significant differences in salivary pH levels of the
subjects between the sampling time points (1st sampling, 6.9 ± 0.5;
2nd sampling, 7.1 ± 0.3; 3rd sampling, 6.9 ± 0.4; 4th sampling,
7.0 ± 0.4; p > 0.05).
3.4. Cotinine levels in saliva samples
Salivary cotinine levels (mean ± SD) of current smokers
(319.4 ± 306.7, ng/ml) were significantly higher than for either non-
smokers (1.4 ± 0.5, ng/ml) or former smokers (1.5 ± 0.5, ng/ml) at
the 4th sampling time point for the whole group (p < 0.05).
3.5. Buccal micronucleus cytome assay results
In this study, 2000 BEC for each sampling, a total of 8000 BEC per
subject and 320000 BEC in total study population have been evaluated
in 4 sampling time points. The frequencies of genotoxicity parameters
observed for each sampling time point are shown in Table 4.
MN frequency was found to be significantly increased in each post-
treatment sampling time points (2nd, 3rd and 4th) when compared to
the pre-treatment sampling time point (1st) in the study population
(Table 4; p < 0.001).
Micronucleated cells frequencies were significantly increased in
each post-treatment sampling time points when compared to the pre-
treatment sampling time point in the study population (Table 4;
p < 0.001). Additionally, micronucleated cell frequencies enumerated
at the 3rd sampling time point were significantly higher than at the 2nd
sampling point in the study population (Table 4; p < 0.05).
The frequency of NBUDs at the 4th sampling time point was found
to be significantly higher than at the 1st sampling point in the study
population (Table 4; p < 0.05).
The BC, CC, KhC, PC, and KC frequencies in pre- and post-treat-
ments did not show a significant difference among the sampling time
points in study population (Table 4; p > 0.05). Frequency of BNC was
significantly elevated in each post-treatment sampling time points (2nd,
3rd and 4th) against pre-treatment sampling time point (1st) in study
population (Table 4; p < 0.05).
In terms of patient smoking habits, MN frequency and micro-
nucleated cell frequency enumerated were significantly lower among
non-smokers (3.0 ± 0.8; 2.6 ± 0.9, respectively) when compared to
either current smokers (5.4 ± 1.6; 4.9 ± 1.6, respectively) or former

22
G. Alp et al.
smokers (4.2 ± 0.9; 3.9 ± 1.0, respectively) (p < 0.001) at the pre-
treatment sampling time point (1st).
The MN frequency (mean ± SD) was not found to be significantly
different between subjects exposed to X-ray (periapical/panoramic)
(7.1 ± 2.8) and non-exposed subjects (7.5 ± 2.8) (p > 0.05) at the
pre-treatment time point (1st sampling).
This study uncovered no significant correlation between the unit
number of PFM-FDPs and MN, micronucleated cell, NBUD, BNC, CC,
PC, or KC frequencies measured (p > 0.05). There was a significant
negative correlation between basal cell frequency and the unit number
of PFM-FDPs at the 2nd and 3rd sampling time points post-treatment
(r = −0.346, p = 0.029; and r = −0.351, p = 0.026, respectively).
There was a significant correlation between KhC frequency and the unit
number of PFM-FDPs at the 2nd sampling time point (r = 0.330,
p = 0.037). There was no significant correlation between MN, micro-
nucleated cell, BC, NBUD, BNC, CC, PC, KC, or KhC frequencies ob-
served and either saliva pH levels or saliva cotinine levels.
The modulation of BEC MN frequencies observed in the pre- and
post- treatments by several independent variables such as sex, age,
smoking status, X-ray exposure, dietary habits, pH and mobile phone
usage time were investigated by linear regression analyses. Multiple
linear regression models were significant only for the 1st sampling time
point. The effect of smoking on the MN frequency 1 st sampling was
found to be statistically significant (p < 0.05).
4. Discussion
In this study, the parameters affecting the genotoxicity, the pro-
liferative potential, and the occurrence of other nuclear anomalies, such
as cytokinetic defects and cell death in exfoliated BEC, in subjects
treated with Ni-Cr based PFM-FDPs were investigated at 4 different
time points. Our analyses of the scientific literature indicate that ours is
the first study to consider PFM-FDPs in this fashion. The BEC and saliva
samples from 40 subjects were obtained during the pre- and post-
treatments of to install between 3 and 15 of PFM-FDPs units. Although
the subjects involved acted as their own controls, they were recruited
homogenously in terms of sex, age, smoking status, and they were
otherwise healthy volunteers with no medical and/or allergy history.
Current smokers, former smokers and nonsmokers were scored via a
questionnaire at the beginning of the study, and volunteer smoker
status was also confirmed at the end of the study through saliva cotinine
measurements. The highest levels of saliva cotinine were detected for
current smokers, decreasing for former smokers and non-smokers, re-
spectively.
The Ni-Cr alloys used in this study are the metallic framework
material most widely used for PFM-FDPs, especially among developing
countries [10,16], as a result of their increased elastic modulus, thermal
compatibility with conventional veneering porcelains [28], good clin-
ical performance [3,4] and low cost. However, these alloys have the
disadvantage of susceptibility to corrosion and the side-products of this
corrosion process have the potential to cause allergic reactions, hy-
persensitivity and other reactions with host tissues [35]. The major
constituents of these alloys are generally nickel (60–84% nickel) and
chromium (8–26% chromium) [14] and in this study, the Ni-Cr alloy
used had 62% Ni and 25% Cr as the major ingredients. As a result, the
properties of the oral environment in which they are situated can fur-
ther contribute to the corrosive properties of alloys; elements could be
released and come into contact with the surrounding soft and hard
tissues [8]. The flow of saliva removes dissolved elements from the
surface of the alloy; therefore, the concentration of the elements that
have accumulated in the adjacent tissues can vary [18]. In many in
vitro studies, it has been reported that the release of Ni and Cr from Ni-
Cr alloy was affected by several factors such as temperature changes
during casting and porcelain firing [36], reuse of the alloy [19], im-
purities [18] and especially, the pH of the corrosive solution was found
to be of great significance [6,11]. Wataha and Lockwood [12]
23
Mutat Res Gen Tox En 827 (2018) 19–26
investigated the elemental release of eight different types of dental
casting Ni-Cr alloys into cell-culture medium over a 10-month period to
interpret the long-term biological risk of these alloys. They reported
that the release of elements continued throughout the 10-month period
and most of the alloys tested reached a constant rate of element release
after 100 days incubation, whereas none of the alloys tested completely
stopped elemental release [12]. Thus, in this study, Ni and Cr elemental
release was measured in artificial saliva at various pH levels, thereby
representing oral environments under different conditions such as
acidic, basic and neutral nature of food intake. In this study, alloys that
had not been reused before installation were used. Our results indicate
that Ni release was highest after 28 days of immersion in an acidic
environment (pH = 2.3). Release of both Ni and Cr in acidic pH were
found to be high for all of the immersion durations used in this study.
The elemental releases were decreased when the acidic pH approached
neutral pH.
While the use of Ni-Cr based dental alloys is commonplace and the
reactions of these alloys with tissue are well documented, their geno-
toxic and cytotoxic effects on adjacent tissues remain unknown due to a
current lack of human based studies, partially motivating this current
study. Although Ni-Cr based dental alloys are not directly evaluated in
the International Agency for Research on Cancer (IARC) monographs,
there are IARC evaluations of both Ni and Cr elements with various
oxidation states, and also for their compounds containing Ni and Cr.
According to such studies, implanted foreign bodies of metallic cobalt,
metallic nickel and an alloy powder containing 66–67% Ni, 13–16% Cr
and 7% Fe has been classified as possibly carcinogenic to humans (IARC
Group 2B) [37], Ni compounds and also Chromium (VI) compounds
have been classified as carcinogenic to humans (IARC Group 1) [38],
metallic Chromium, Chromium (III) compounds (IARC Group 3) [17],
and Chromium based alloys were not classifiable by risk of carcino-
genicity posed to humans (IARC Group 3) [37]. Use of genotoxicity
endpoints as early effect biomarkers in molecular epidemiology studies
are quite informative both for gauging the extent of DNA damage and
for predictions of future cancer development. Since there is a lack of
concrete data concerning the genotoxicity of Ni-Cr based PFM-FDPs
under real life conditions, the data in this study are will be very useful
for further evaluations of these effects on the target tissue. In the cur-
rent study, the biocompatibility of the Ni and Cr in the alloys, but not
the ceramics, were the focus, as the inertness of feldspathic ceramics
had already been proved in previous studies [10].
In previous studies, the metal ions released were found in tissues
adjacent to the dental cast alloys in high concentrations [39]. Hence the
BEC, acting as the first barrier for proximate carcinogens [40], being
located within the contact area of the PFM-FDPs and exposed to the
corrosive substances, were chosen for study. Therefore, they represent a
target tissue for genomic instability events that are induced by the
metal ions released [25], and thus used as a target tissue of the study
population for FDPs and as a possible non-invasive diagnostic method
[41–43].
MN is a genomic instability biomarker [44] where portions of the
genome remain unincorporated during the transition from metaphase-
anaphase of the division of the mitotic nucleus in the basal layers of the
epithelium [43] and it manifests as encapsulated extra-nuclear bodies
in daughter cells that originate from either whole chromosomes or
chromosome fragments [32]. The maximal rate of MN formation de-
pends on the DNA damage on the basal cell turnover rate and seen
between 1 and 3 weeks after the exposure [32,43].
Recent meta-analysis results have suggested that MN frequency in
BEC can be used in prescreening and in the follow up of pre-cancerous
oral lesions [45].
BMCyt assay is a preferred method among dentistry applications
[46–52], however none of these studies cited here considered Ni-Cr
PFM-FDPs. In the scientific literature, the study of Baricevic et al. [21]
is the only in vivo human genotoxicity study concerning the use of Ni-
Cr alloys used in fixed prosthodontic appliances. However, in contrast
G. Alp et al.
to our study, the Comet Assay was used in analyses of BEC cells and the
study population comprised subjects wearing Ni-Cr based fixed
(n = 19) or Co-Cr-Mo based removable (n = 11) prosthodontic appli-
ances for longer than 5 years. These test subjects were compared to the
edentulous control group without prosthodontic appliances (n = 25)
[21]. The study reported DNA damage, suggesting the elemental release
of metals after long-term use [21]. Therefore, the results of this study
are important and original in design in the determining the likely
genotoxicity of Ni-Cr based PFM-FDPs in BEC.
In this study, the frequencies of MN before the operative procedures
(1st sampling) indicated that normal individual range values for
healthy adults and termed this as the spontaneous MN frequency. The
1st sampling point BMCyt parameters of the present study were com-
pared to the study of Thomas et al. [34], which evaluated healthy
adults (who were older than our study cohort) for their spontaneous
parameters. Spontaneous MN frequencies among healthy adults
(1.4 ± 1.6) [34] were lower than for the subjects in the current study
(4.0 ± 1.5). These differences may be attributable to inter-individual
variability, the differences in the test methods used and also scorers.
In Turkish population studies, the baseline MN frequencies were
found to be between 1.0–4.4 per thousand cells [53–58] while the
present study has baseline BEC MN frequency of 4.0‰ ± 1.5. How-
ever, studies have variations according to the study population (i.e. age,
sex), region, laboratory and methodologies utilized (i.e. cell numbers
counted, staining, scorer). As reported in Bolognesi et al. [45,59]; MN
scoring is not yet a standardized procedure and the identification of
different cell types and nuclear anomalies differs from each laboratory,
depending on the study population, the pool of experience available in
the laboratories and scoring criteria applied.
In the present study, the MN parameters were significantly higher in
all post-treatment time points, suggesting that Ni-Cr based PFM-FDPs
contributed to the genotoxicity observed.
It has been suggested that cytome approach may improve the pre-
dictive value of the MN assay in BEC [45]. The NBUD frequency as a
gene amplification biomarker [60] was significantly higher only at the
4th sampling time point after the delivery of the restoration, which
could be interpreted as the genotoxic effect of the material. Ad-
ditionally, the frequency of BNC can act as an indicator of failed cy-
tokinesis [61] and was found to be significantly elevated after the re-
storation delivery. The frequencies of nuclear alterations that indicate
cell death (condensed chromatin, karyorrhexis, pyknotic, and kar-
yolytic cell frequencies) [34] were not increased after the restoration
delivery. These results suggest that Ni-Cr alloy did not induce cytotoxic
effects at any of the sampling time points. In vitro studies indicate that
there are cytotoxicity results in a three-dimensional (3D) human-de-
rived oral mucosal model for Ni––Cr and Co-Cr base metal alloys [20]
and for reused alloys, including Ni-Cr [19]. However, apart from the
shared emphases on Ni-Cr based alloys, those studies are not compar-
able to this current study in terms of study type, cell type, application,
methodology, physiological conditions and alloy properties.
In the present study, confounding factors such as diet, X-ray ex-
posure, and mobile phone usage for inducing the genotoxic effect were
queried by questionnaire at each sampling time point, and there was no
change identified in these potential confounding values through all
sampling times. It ought be kept in mind, however, that the reliability
of non-validated questionnaires is limited when each of the likely
confounding factors had not been experimentally verified.
Specifically with respect to X-ray exposure, the 1st sampling was
carried out at least 21 days subsequent to X-ray exposure in order to
give sufficient time for BEC turnover [32] to occur so as to prevent
additional genotoxic or cytotoxic effects. In the statistical evaluation,
there was no significant difference in MN frequency between test sub-
jects exposed to X-rays and non-exposed subjects. Finally, any possible
effects due to the confounding factors were controlled using regression
analysis, which showed that they had no effect on MN frequency.
Previous studies that evaluated the genotoxic effects of X-ray exposure
24
Mutat Res Gen Tox En 827 (2018) 19–26
evaluated oral mucosa cells using micronucleus assay. In keeping with
the results of the current study, this former studies found no significant
difference in MN frequency in the same patients before and subsequent
to exposure to X-ray where the interval between the samplings was
10 days [48,49]. Cotinine is one of the major metabolites of nicotine
and is used to quantify exposure to tobacco smoke [62]. In the present
study, we had the opportunity to confirm the information provided on
the questionnaire concerning smoking using salivary cotinine levels in
as a quantitative measure − the saliva cotinine levels among smokers
were shown to be higher than that of the non-smokers. Although we
found higher saliva cotinine levels in current smokers, along with
higher MN frequency, we found no significant correlation between the
saliva cotinine level and MN frequencies and those of other cellular
aberrations.
This study as a human based clinical study revealed that in the
target tissue of subjects, the post-treatment time points subsequent to
Ni-Cr PFM-FDP application showed clear indications of genotoxicity,
but not cytotoxicity. Although avoiding individual variations, con-
ducting time based controlled study and originality in the scientific
literature was achieved, there were a few limitations of this study.
These are; the released ion levels in adjacent tissues were not evaluated
and the follow up period was only three months.
5. Conclusions
This study underscores the importance of the BMCyt-assay as a
biomarker with which to determine the genotoxic and cytotoxic effects
of Ni-Cr alloys. In conclusion, clinicians should use not only those alloys
that exhibit the lowest element releases, and to be aware of the likely
effects of such alloys in both short and long-term usage, but also ought
to inform patients about aspects of their diet and life style conditions
that can affect the amount of elemental release from alloys used in
prosthesis.
Funding
This work was a doctoral thesis that was financially supported by
the Gazi University Research Council [03/2012-13].
Conflict of interest
None.
Acknowledgements
We would like to thank Ankara University Earth Sciences
Application and Research Center (YEBIM) for allowing us to use their
laboratory and equipment. Mehmet Simsek (Ozel Burak Dis Protez
Laboratuari) for preparing Ni-Cr specimens, Huseyin Guler (Ozel Guler
Dis Protez Laboratuari) for preparing PFM-FDPs, Esra Emerce (Gazi
University, Faculty of Pharmacy, Ankara) for the help with saliva co-
tinine analysis, Istatistik Dunyasi and Ahmet Gul for performing the
statistical analyses for this study, and Mustafa Erdem (Gazi University,
Academic Writing and Research Center) for proofreading the paper.
The authors also would like to thank Prof. Dr. Sema Burgaz (Gazi
University, Faculty of Pharmacy) for their critical analysis and com-
ments on the manuscript.
References
[1] Y. Lu, W. Chen, W. Ke, S. Wu, Nickel-based (Ni–Cr and Ni–Cr–Be) alloys used in
dental restorations may be a potential cause for immune-mediated hypersensitivity,
Med. Hypotheses 73 (2009) 716–717.
[2] K.J. Anusavice, Standardizing failure success, and survival decisions in clinical
studies of ceramic and metal-ceramic fixed dental prostheses, Dent. Mater. 28
(2012) 102–111.
[3] T.R. Walton, An up to 15-year longitudinal study of 515 metal-ceramic FDPs: part 2.
G. Alp et al.
Modes of failure and influence of various clinical characteristics, Int. J.
Prosthodont. 16 (2003) 177–182.
[4] H.D. Backer, G.V. Maele, N.D. Moor, L.V. Berghe, Long-term results of short-span
versus long-span fixed dental prostheses: an up to 20-year retrospective study, Int.
J. Prosthodont. 21 (2008) 75–85.
[5] J.C. Wataha, Alloys for prosthodontic restorations, J. Prosthet. Dent. 87 (2002)
351–363.
[6] G. Bayramoglu, T. Alemdaroglu, S. Kedici, A.A. Aksut, The effect of pH on the
corrosion of dental metal alloys, J. Oral Rehabil. 27 (2000) 563–575.
[7] S.P. Kedici, A.A. Aksut, M.A. Kilicarslan, G. Bayramoglu, K. Gokdemir, Corrosion
behaviour of dental metals and alloys in different media, J. Oral Rehabil. 25 (1998)
800–808.
[8] J.C. Wataha, Biocompatibility of dental casting alloys: a review, J. Prosthet. Dent.
83 (2000) 223–234.
[9] H. Bilhan, T. Bilgin, A.F. Cakir, B. Yuksel, J.A. Von Fraunhofer, The effect of mu-
cine, IgA, urea, and lysozyme on the corrosion behavior of various non-precious
dental alloys and pure titanium in artificial saliva, J. Biomater. Appl. 22 (2007)
197–221.
[10] W. Elshahawy, I. Watanabe, M. Koike, Elemental ion release from four different
fixed prosthodontic materials, Dent. Mater. 25 (2009) 976–981.
[11] S. Denizoglu, Z.Y. Duymus, S. Akyalçin, Evaluation of ion release from two base-
metal alloys at various pH levels, J. Int. Med. Res. 32 (2004) 33–38.
[12] J.C. Wataha, P.E. Lockwood, Release of elements from dental casting alloys into cell
culture medium over 10 months, Dent. Mater. 14 (1998) 158–163.
[13] F. Faccioni, P. Franceschetti, M. Cerpelloni, M.E. Fracasso, In vivo study on metal
release from fixed orthodontic appliances and DNA damage in oral mucosa cells,
Am. J. Orthod. Dentofacial. 124 (2003) 687–694.
[14] S.B. Jones, R.L. Taylor, J.S. Colligon, D. Johnson, Effect of element concentration on
nickel release from dental alloys using a novel ion beam method, Dent. Mater. 26
(2010) 249–256.
[15] W. Geurtsen, Biocompatibility of dental casting alloy, Crit. Rev. Oral Biol. Med. 13
(2002) 71–84.
[16] C. Zhihong, L. Yezhen, G. Zhiyuan, Y. Zhongqiao, L. Xiaoxue, R. Yifeng, F. Weikun,
H. Jiliang, Comparison of the cytogenotoxicity induced by five different dental
alloys using four in vitro assays, Dent. Mater. J. 30 (2011) 861–868.
[17] IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, Chromium,
Nickel and Welding 49 International Agency for Research on Cancer Lyon, France,
1990.
[18] J.F. Lopez-Alıas, J. Martinez-Gomis, J.M. Anglada, M. Peraire, Ion release from
dental casting alloys as assessed by a continuous flow system: nutritional and tox-
icological implications, Dent. Mater. 22 (2006) 832–837.
[19] A.S. Al-Hiyasat, H. Darmani, The effects of recasting on the cytotoxicity of base
metal alloys, J. Prosthet. Dent. 93 (2005) 158–163.
[20] E.L. McGinley, G.P. Moran, G.J.P. Fleming, Biocompatibility effects of indirect ex-
posure of base-metal dental casting alloys to a human-derived three-dimensional
oral mucosal model, J. Dent. 41 (2013) 1091–1100.
[21] M. Baricevic, I. Ratkaj, M. Mladinic, D. Zeljezic, S.P. Kraljevic, B. Loncar,
M.M. Stipetic, In vivo assessment of DNA damage induced in oral mucosa cells by
fixed and removable metal prosthodontic appliances, Clin. Oral Invest. 16 (2012)
325–331.
[22] S.C. Tripuraneni, S.K. Namburi, Evaluation of genotoxicity of recycled Ni-Cr dental
casting alloys: an in vitro study, J. Appl. Biomater. Biomech. 6 (2008) 47–54.
[23] S. Burgaz, G. Çakmak, O. Erdem, M. Yilmaz, A.E. Karakaya, Micronuclei frequencies
in exfoliated nasal mucosa cells from pathology and anatomy laboratory workers
exposed to formaldehyde, Neoplasma 48 (2001) 144–147.
[24] S. Burgaz, G.C. Demircigil, M. Yılmazer, N. Ertas, Y. Kemaloglu, Y. Burgaz,
Assessment of cytogenetic damage in lymphocytes and in exfoliated nasal cells of
dental laboratory technicians exposed to chromium cobalt, and nickel, Mutat. Res.
521 (2002) 47–56.
[25] N. Holland, C. Bolognesi, M. Kirsch-Volders, S. Bonassi, E. Zeiger, S. Knasmueller,
M. Fenech, The micronucleus assay in human buccal cells as a tool for biomoni-
toring DNA damage: the HUMN project perspective on current status and knowl-
edge gaps, Mutat. Res. 659 (2008) 93–108.
[26] S. Knasmueller, N. Holland, G. Wultsch, B. Jandl, S. Burgaz, M. Misik, A. Nersesyan,
Use of nasal cells in micronucleus assays and other genotoxicity studies,
Mutagenesis 26 (2011) 231–238.
[27] M. Fenech, Cytokinesis-block micronucleus assay evolves into a cytome assay of
chromosomal instability, mitotic dysfunction and cell death, Mutat. Res. 600 (2006)
58–66.
[28] C.M. Wylie, R.M. Shelton, G.J.P. Fleming, A.J. Davenport, Corrosion of nickel-based
dental casting alloys, Dent. Mater. 23 (2007) 714–723.
[29] ISO 10271 Standard, Dental Metallic Materials—Corrosion Test Methods, 1st ed.,
ISO, Geneva, Switzerland, 2001.
[30] V. Binnie, S. McHugh, L. Macpherson, B. Borland, K. Moir, K. Malik, The validation
of self-reported smoking statusby analysing cotinine levels in stimulated and un-
stimulated saliva, serum and urine, Oral Dis. 10 (2004) 287–293.
[31] S. Chiappin, G. Antonelli, R. Gatti, E.F. De Palo, Saliva specimen: a new laboratory
tool for diagnostic and basic investigation, Clin. Chim. Acta 383 (2007) 30–40.
[32] M. Fenech, N. Holland, W.P. Chang, E. Zeiger, S. Bonassi, The Human MicroNucleus
Project—an international collaborative study on the use of the micronucleus tech-
nique for measuring DNA damage in humans, Mutat. Res. 428 (1999) 271–283.
[33] S. Muchandi, H. Walimbe, M.N.A. Bijle, M. Nankar, S. Chaturvedi, P. Karekar,
Comparative evaluation and correlation of salivary total antioxidant capacity and
salivary pH in caries-free and severe early childhood caries children, J. Contemp.
Dent. Pract. 16 (2015) 234–237.
[34] P. Thomas, N. Holland, C. Bolognesi, M. Kirsch-Volders, S. Bonassi, E. Zeiger,
25
Mutat Res Gen Tox En 827 (2018) 19–26
S. Knasmueller, M. Fenech, Buccal micronucleus cytome assay, Nat. Protoc. 4
(2009) 825–837.
[35] J.C. Setcos, A. Babaei-Mahani, L.D. Silvio, I.A. Mjor, N.H.F. Wilson, The safety of
nickel containing dental alloys, Dent. Mater. 22 (2006) 1163–1168.
[36] M. Anwar, A. Tripathi, S.K. Kar, K.C. Sekhar, Effect of PFM firing cycles on the
mechanical properties phase composition, and microstructure of Nickel-Chromium
alloy, J. Prosthodont. 00 (2015) 1–8.
[37] IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, Surgical
Implants and Other Foreign Bodies 74 International Agency for Research on Cancer
Lyon, France, 1999.
[38] IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, Arsenic,
Metals, Fibres, and Dusts 100C International Agency for Research on Cancer Lyon,
France, 2012 (sup 7, 49).
[39] P. Garhammer, G. Schmalz, K.A. Hiller, T. Reitinger, Metal content of biopsies
adjacent to dental cast alloys, Clin. Oral Invest. 7 (2003) 92–97.
[40] B. Kashyap, P.S. Reddy, Micronuclei assay of exfoliated oral buccal cells: means to
assess the nuclear abnormalities in different diseases, J. Canc. Res. Ther. 8 (2012)
184–191.
[41] S. Bonassi, E. Coskun, M. Ceppi, C. Lando, C. Bolognesi, S. Burgaz, N. Holland,
M. Kirsh-Volders, S. Knasmueller, E. Zeiger, D. Carnesoltas, D. Cavallo, J. da Silva,
V.M. Andrade, G.C. Demircigil, A.D. Odio, H. Donmez-Altuntas, G. Gattas, A. Giri,
S. Giri, B. Gomez-Meda, S. Gomez-Arroyo, V. Hadjidekova, A. Haveric, M. Kamboj,
K. Kurteshi, M.G. Martino-Roth, R.M. Montoya, A. Nersesyan, S. Pastor-Benito,
D.M.F. Salvadori, A. Shaposhnikova, H. Stopper, P. Thomas, O. Torres-Bugarın,
A.S. Yadav, G.Z. Gonzalez, M. Fenech, The HUman MicroNucleus project on
eXfoLiated buccal cells (HUMNXL): the role of life-style, host factors, occupational
exposures, health status, and assay protocol, Mutat. Res. 728 (2011) 88–97.
[42] M. Fenech, N. Holland, E. Zeiger, W.P. Chang, S. Burgaz, P. Thomas, C. Bolognesi,
S. Knasmueller, M. Kirsch-Volders, S. Bonassi, The HUMN and HUMNxL interna-
tional collaboration projects on human micronucleus assays in lymphocytes and
buccal cells—past, present and future, Mutagenesis 26 (2011) 239–245.
[43] M.P. Rosin, The use of the micronucleus test on exfoliated cells to identify anti-
clastogenic action in humans: a biological marker for the efficacy of chemopre-
ventive agents, Mutat. Res. 267 (1992) 265–276.
[44] V.S. Dhillon, P. Thomas, M. Fenech, Comparison of DNA damage and repair fol-
lowing radiation challenge in buccal cells and lymphocytes using single-cell gel
electrophoresis, Int. J. Radiat. Biol. 80 (2004) 517–528.
[45] C. Bolognesi, S. Knasmueller, A. Nersesyan, P. Roggieri, M. Ceppi, M. Bruzzone,
E. Blaszczyk, D. Mielzynska-Svach, M. Milic, S. Bonassi, D. Benedetti, J.D. Silva,
R. Toledo, D.M.F. Salvadori, H.G. Restrepo, M. Filipic, K. Hercog, A. Aktas,
S. Burgaz, M. Kundi, T. Grummt, P. Thomas, M. Hor, M. Escudero-Fung, N. Holland,
M. Fenech, Inter-laboratory consistency and variability in the buccal micronucleus
cytome assay depends on biomarker scored and laboratory experience: results from
the HUMNxl international inter-laboratory scoring exercise, Mutagenesis 00 (2016)
1–10.
[46] D.A. Ribeiro, G. Oliveira, G.M. Castro, F. Angelieri, Cytogenetic biomonitoring in
patients exposed to dental X-rays: comparison between adults and children,
Dentomaxillofac. Radiol. 37 (2008) 404–407.
[47] F. Angelieri, G.R. Oliveira, E.K. Sannomiya, D.A. Ribeiro, DNA damage and cellular
death in oral mucosa cells of children who have undergone panoramic dental
radiography, Pediatr. Radiol. 37 (2007) 561–565.
[48] F. Angelieri, T.C.G. Moleirinho, V. Carlin, C.T.F. Oshima, D.A. Ribeiro,
Biomonitoring of oral epithelial cells in smokersand non-smokers submitted to
panoramic X-ray: comparison between buccal mucosa and lateral border of the
tongue, Clin. Oral Invest. 14 (2010) 669–674.
[49] E.M.M. Cerqueira, I.S. Gomes-Filho, S. Trindade, M.A. Lopes, J.S. Passos,
G.M. Machado-Santelli, Genetic damage in exfoliated cells from oral mucosa of
individuals exposed to X-rays during panoramic dental radiographies, Mutat. Res.
562 (2004) 111–117.
[50] G. Visalli, B. Baluce, S.L. Maestra, R.T. Micale, L. Cingano, S.D. Flora, A.D. Pietro,
Genotoxic damage in the oral mucosa cells of subjects carrying restorative dental
fillings, Arch. Toxicol. 87 (2013) 179–187.
[51] F. Camacho-Alonso, M. Sanchez-Siles, O.G. Aguila, No evidence of genotoxic da-
mage in a group of patients with titanium dental implants and different metal re-
storations in the oral cavity, Clin. Implant Dent. Relat. Res. 17 (2015) 811–821.
[52] F. Angelieri, V. Carlin, R.A. Martins, D.A. Ribeiro, Biomonitoring of mutagenicity
and cytotoxicity in patients undergoing fixed orthodontic therapy, Am. J. Orthod.
Dentofac. 139 (2011) 399–404.
[53] B. Karahalil, E. Kadioglu, A.M. Tuzuner-Oncul, E. Cimen, E. Emerce, R.S. Kisnisci,
Micronucleus assay assessment of possible genotoxic effects in patients treated with
titanium alloy endosseous implantsor miniplates, Mutat. Res. Genet. Toxicol.
Environ. Mutagen. 760 (2014) 70–72.
[54] E. Eker Derici, H. Koyuncu, N.Ö. Şahin, A. Yüksel, M. Berköz, S. Diler Budak,
S. Akgül Altan, Determination of genotoxic effects of hookah smoking by micro-
nucleus and chromosome aberration methods, Med. Sci. Monit. 22 (2016)
4490–4494.
[55] O.S. Aslantürk, T. Çelik Askin, Genotoxic risk assessment in professionals working
hairdressers area using buccal micronucleus assay, in Aydın City, Turkey, Environ.
Sci. Pollut. Res. Int. 29 (2017) (Epub ahead of print).
[56] A. Celik, S. Yildirim, S.Y. Ekinci, B. Tasdelen, Bio-monitoring for the genotoxic
assessment in road construction workers as determined by the buccal micronucleus
cytome assay, Ecotoxicol. Environ. Saf. 92 (2013) 265–270.
[57] C. Saatci, Y. Ozkul, S. Tahiri, A.O. Caglayan, A.B. Turhan, M. Dundar, The Effect of
maras powder on DNA methylation and micronucleus formation in human buccal
tissue, J. Toxicol. Environ. Health A 71 (2008) 396–404.
[58] A. Saruhanoglu, S. Ergun, M. Kaya, S. Warnakulasuriya, M. Erbagcı, S. Ozturk,
G. Alp et al.
E. Deniz, S. Ozel, K. Cefle, S. Palanduz, H. Tanyeri, Evaluation of micronuclear
frequencies in both circulating lymphocytes and buccal epithelial cells of patients
with oral lichen planus and oral lichenoid contact reactions, Oral Dis. 20 (2014)
521–527.
[59] C. Bolognesi, S. Bonassi, S. Knasmueller, M. Fenech, M. Bruzzone, C. Lando,
M. Ceppi, Clinical application of micronucleus test in exfoliated buccal cells: a
systematic review and metanalysis, Mutat. Res. Rev. Mutat. Res. 766 (2015) 20–31.
[60] M. Fenech, J.W. Crott, Micronuclei, nucleoplasmic bridges and nuclear buds in-
duced in folic acid deficient human lymphocytes-evidence for breakage-fusion-
26
Mutat Res Gen Tox En 827 (2018) 19–26
bridge cycles in the cytokinesis-block micronucleus assay, Mutat. Res. 504 (2002)
131–136.
[61] P. Thomas, S. Harvey, T. Gruner, M. Fenech, The buccal cytome and micronucleus
frequency is substantially altered in Down’s syndrome and normal ageing com-
paredto young healthy controls, Mutat. Res. 638 (2008) 37–47.
[62] P. Dhar, Measuring tobacco smoke exposure: quantifying nicotine/cotinine con-
centration in biological samples by colorimetry, chromatography and immunoassay
methods, J. Pharm. Biomed. Anal. 35 (2004) 155–168.