Environmental Toxicology and Pharmacology 66 (2019) 104–108

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Environmental Toxicology and Pharmacology

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

Evaluation of genetic polymorphisms in MMP2, MMP9 and MMP20 in T

Brazilian children with dental fluorosis
Priscilla Coutinho Romualdoa, Carolina Maschietto Pucinellia, Patricia Nivoloni Tannureb,
Paulo Nelson-Filhoa, Raquel Assed Bezerra Segatoa, João Armando Brancherc,
Nilza Letícia Magalhãesa, Marcelo de Castro Costad, Lívia Azeredo A. Antunese,
Leonardo Santos Antunese, Marília Afonso Rabelo Buzalaff, Senda Charonef,
⁎
Erika Calvano Küchlera,
a
Department of Pediatric Dentistry, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, Brazil
b
School of Dentistry, Veiga de Almeida University, Rio de Janeiro, RJ, Brazil
c
Department of Dentistry, Universidade Positivo, Curitiba, Brazil
d
Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
e
School of Dentistry, Fluminense Federal University, Nova Friburgo, RJ, Brazil
f
Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Recent studies suggested that genetics contribute to differences in dental fluorosis (DF) susceptibility among
Fluorosis individuals having the same environmental exposure. This study evaluated if MMP2, MMP9 and MMP20 are
Dental expressed during enamel development and assessed the association between polymorphisms in these genes with
Immunohistochemistry DF. Mice susceptible and resistant to DF were used to evaluate if MMPs were candidate genes for DF. The animals
Genetic susceptibility
received fluoride and their enamels were used for immunohistochemistry. Additionally, 481 subjects from a city
Matrix metalloproteinases
Polymorphisms
with fluoridation of public water supplies were recruited. Genotyping was performed using real time PCR.
SNPs Allele/genotype frequencies were compared between groups. MMP2, MMP9 and MMP20 immunostaining was
detected in both animal groups. DF was observed in 22.4% of the subjects. A borderline association was observed
in MMP2 (rs243865), MMP9 (rs17576) and in MMP20 (rs1784418) (p = 0.06, p = 0.08 and p = 0.06 respec-
tively). Briefly, MMPs were expressed during enamel maturation and genetic polymorphisms were not associated
with DF.

1. Introduction hydrolyzed by enamel matrix proteinases (Robinson et al., 1998).

Dental fluorosis (DF) occurs when enamel is formed in the presence
of high levels of systemic fluoride and is endemic in at least 25 coun-
tries worldwide. Consumption of excess fluoride through water can lead
to DF (Pramanik and Saha, 2017). Although DF is considered as a defect
of enamel maturation, some studies have shown that this type of
fluorosis is more severe when the exposure to fluoride happens in both
secretory and maturation levels of enamel formation. Teeth formed in
this condition have an increased porosity in enamel (Denbesten and Li,
2011; Zhang et al., 2014). Besides, the mineral content of fluorosed
enamel is reduced, and the enamel contains greater levels of proteins
such as amelogenin (DenBesten, 1999).
Amelogenins comprise 90% of the enamel organic matrix and are
Fluoride induced changes in proteinase activity that could result in a
delay in the removal of amelogenins, with a subsequent delayed enamel
matrix mineralization. It is known that amelogenins and enamel matrix
proteinases are synthesized by ameloblasts (Zhang et al., 2007). Ame-
loblasts are responsible for enamel formation that occurs in stages,
depending on the ameloblasts morphology. Ameloblasts secrete enamel
proteins and enamel matrix proteases, such as matrix metalloprotei-
nases (MMPs) (Bartlett, 2013; Llano et al., 1997; Simmer et al., 2012).
MMPs are members of an enzyme family, which require a zinc ion in
their active site for catalytic activity. Some studies have demonstrated
the expression of MMP2, MMP9 and MMP20 during enamel formation
(Bartlett, 2013; Caron et al., 2001; Heikinheimo and Salo, 1995; Llano
et al., 1997; Simmer et al., 2012). MMP2 and MMP9 are gelatinases

⁎
Corresponding author at: Department of Pediatric Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo – USP, Avenida do Café s/n, Monte
Alegre, 14040-904, Ribeirão Preto, SP, Brazil.
E-mail address: erika.kuchler@up.edu.br (E.C. Küchler).

https://doi.org/10.1016/j.etap.2018.12.016
Received 8 March 2018; Received in revised form 19 December 2018; Accepted 20 December 2018
Available online 21 December 2018
1382-6689/ © 2018 Published by Elsevier B.V.

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P.C. Romualdo et al.
with collagen-degrading ability and have important roles in tooth for-
mation and mineralization (Niu et al., 2011). MMP20 is an enamelisin,
and is responsible for the initial hydrolysis of amelogenins and pro-
motes appositional growth until the enamel layer reaches the entire
thickness (Bartlett, 2013; Llano et al., 1997; Simmer et al., 2012). Since
MMP20 is essential for proper dental enamel formation, any alteration
in MMP20 expression or activity by fluoride is likely to affect enamel
formation (Sharma et al., 2011; Zhang et al., 2007). However, it was
demonstrated that enamel fluorosis cannot be solely attributed to a
reduction of MMP20 expression (Sharma et al., 2011).
In the last decade, the study of the genetic component of DF has
been emerging. The genetic susceptibility to DF and the association
between fluorosis and genetic polymorphisms has been the subject of
several studies, both in human populations (Escobar-Garcia et al., 2016;
Jiao et al., 2013; Kuchler et al., 2017a, b; Wen et al., 2012) and animal
models, in which a mouse strain susceptible to DF (A/J) and a mice
strain resistant to dental fluorosis (129P3/J) are studied (Everett, 2011;
Everett et al., 2002, 2009; Everett et al., 2011). A recent review
(Pramanik and Saha, 2017) reported that genetic variants in some
candidate genes could increase or decrease the risk of DF among the
exposed individuals in endemic areas in different studies (Huang et al.,
2008; Wen et al., 2012; Jiao et al., 2013; Zhang et al., 2015; Küchler
et al., 2017b). However, no study has investigated polymorphisms in
MMP2, MMP9 and MMP20 in DF.
Therefore, this study aimed to explore the association between
MMPs and DF evaluating if MMP2, MMP9 and MMP20 are expressed
during mouse enamel development and evaluating the association be-
tween genetic polymorphisms in these genes with human DF.
2. Materials and methods
2.1. Expression of MMP2, MMP9 and MMP20 during enamel maturation
2.1.1. Animals and treatment
The experimental protocols for the animals were approved by the
Ethics Committee for Animal Experiments of the Bauru Dental School,
University of São Paulo, Brazil (protocol No. 08/20113).
Animal models susceptible and resistant to DF were used in this
study to evaluate if MMP2, MMP9 and MMP20 are candidate genes for
DF. Therefore, 3 susceptible mice (A/J) and 3 resistant mice (129P3/J),
3 weeks old (after being weaned), were used. All animals were housed
in pairs in metabolic cages to allow the measurement of their water
intake. The animals received drinking water containing 50 mg F/l (as
NaF) for 6 weeks as established by a previous study that evaluated the
proteins involved in the enamel development of these mice (Charone
et al., 2016). Since susceptible mice tend to consume more water than
resistant mice, the fluoride concentrations in the water given to the
susceptible mice were adjusted weekly to equalize the fluoride intake
by the 2 strains [Carvalho et al., 2009]. At the end of the treatment
period, mice were anesthetized with ketamine/xylazine and dental ar-
ches were collected for the histotechnical processing and identification/
localization of MMP2, MMP9 and MMP20 by Immunohistochemistry.
The incisors were selected for the study due the fact that the incisor
teeth of rodents grow continuously, thus, after 6 weeks of fluiride
treatment, the evaluated incisor fully developed (including amelogen-
esis) under fluoride treatment.
2.1.2. Histotechnical processing and immunohistochemistry – expression of
matrix metalloproteinases
The dental arches were fixed by immersion in 10% buffered for-
maldehyde for 24 h and then washed for 4 h in running water. The
dental arches were kept in EDTA-based solution until complete demi-
neralization. After this, the samples were washed in running water for
2 h, dehydrated in increasing concentrations of alcohol, diaphanized in
xylol and embedded in paraffin. The blocks with were cut using a mi-
crotome (Leica RM2145; Leica Microsystems GmbH, Wetzlar,
105
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Environmental Toxicology and Pharmacology 66 (2019) 104–108
Germany) and five-micrometer-thick sections were obtained.
The immunohistochemical analysis was performed for the identifi-
cation of matrix metalloproteinases (MMP2, MMP9, and MMP20) in
representative sections of each group. The reactions were performed
using the avidin-biotin-peroxidase complex (indirect method), as de-
scribed previously (da Silva et al., 2012). The following primary anti-
bodies were used: anti-MMP2 (polyclonal goat antibody sc-8835, 1:200
dilution; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-MMP9
(polyclonal goat antibody sc-6840; Santa Cruz Biotechnology Inc.;
1:200 dilution) and anti-MMP20 (polyclonal goat antibody sc-26926;
Santa Cruz Biotechnology Inc.; 1:200 dilution). The slides were in-
cubated with biotinylated secondary antibody (rabbit anti-goat IgG-
HRP: sc-2768; Santa Cruz Biotechnology Inc., 1:200 dilution). Negative
controls were dental arches sections treated with phosphate saline
buffer instead of the specific antibodies.
The identification of MMPs was performed under reflected light
using the Axio Imager.M1 (Zeiss) microscope. Results were expressed in
a qualitative manner, considering the presence/absence and localiza-
tion of immunostaining, as described previously (da Silva et al., 2012).
2.2. Human population
The local Human Ethics Committee (113/09) approved this study.
Informed consent was obtained from all participating individuals or
parents/legal guardians.
2.2.1. Type of study and sampling
This cross-sectional eligible unrelated healthy subjects from 6 to 18
years of age recruited at the Pediatric Dental Clinics, Federal University
of Rio de Janeiro. This population lived in Rio de Janeiro, a city with
fluoridation of public water supplies in which the fluoride levels ranged
from 0.10 to 0.55 ppm F (parts per million of fluoride). The subjects
were divided in groups according presence or absence of DF. The cur-
rent analysis used data from early-erupting permanent teeth. For the
mixed dentition, only erupted permanent teeth were assessed. This
sample collection and description was previously described in Küchler
et al. (2017a).
2.2.2. Determination of dental fluorosis phenotype
Trained examiners conducted dental examinations (Kuchler et al.,
2017a). Subjects were seated in a dental chair, and the examiner used a
probe and dental mirror according to the criteria recommended by the
World Health Organization guidelines. DF was assessed using the
Dean´s index modified (Rozier, 1994) with the examination and a score
was registered. This index allows the classification of fluorosis into
three degrees: mild (very mild and mild), moderate, and severe. The
questionable degree was excluded. The current analysis used data from
early-erupting permanent teeth and did not use DF phenotype data
from primary dentition. For the mixed dentition, only erupted perma-
nent teeth were assessed.
2.2.3. DNA samples and genotyping
Genomic DNA for molecular analysis was extracted from buccal
cells based on the reported method (Kuchler et al., 2012). Five genetic
polymorphisms in 3 MMPs were genotyped by real time polymerase
chain reactions (Real Time PCR) using the Taqman assay. The selected
genes are expressed on the enamel development (http://bite-it.helsinki.
fi/). The characteristics of the studied polymorphisms are presented in
Table 1.
2.2.4. Statistical analysis
The data were analyzed using the Epi Info 3.5.7. T test, chi-square
test and odds ratio calculations were used to compare age and sex be-
tween DF affected and DF free subjects. Chi-square or Fisher´s exact
tests and odds ratios were used to compare allele and genotype dis-
tributions between ‘DF’ and ‘DF free’ groups in an additive and
P.C. Romualdo et al.
Table 1
Candidate studied genes and genetic polymorphisms.
Gene Genetic polymorphism Locus
MMP2 rs243865 16q13
rs243867
MMP9 rs17576 20q1-q13
MMP20 rs1784418 11q22.2
rs1711437
Note: Bold form indicates ancestral allele; *change in transcription; reported regulation of gene expression determined by the mentioned allele **missense; MAF
means minor allele frequency obtained from databases: http://www.ncbi.nlm.nih.gov.
dominant model. Logistic regression analysis was also implemented for
all polymorphisms analysed using ethnicity as covariate in order to test
the possibility of ethnicity background influence. A level of significance
of 0.05 was used.
Hardy-Weinberg equilibrium was evaluated using the chi-square
test within each polymorphism.
3. Results
3.1. Animal immunohistochemistry
In the animal, Fig. 1 demonstrates the immunohistochemical find-
ings for MMP2, MMP9 and MMP20 in the susceptible and resistant
mice. This revealed that, during enamel development, im-
munopositivity for MMP2, MMP9 and MMP20 was present and could be
observed in the enamel matrix near to the dental papilla. MMP2, MMP9
and MMP20 were moderately stained in ameloblast.
The immunostainings of MMP2, MMP9 and MMP20 were observed
in mice from the both groups, susceptible and resistant to DF, conse-
quently, these MMPs were considered candidate genes for DF.
3.2. Human population genotyping
Hundred eight subjects presented DF and 373 were DF free subjects.
Mild DF affected 85 subjects (78.7%), moderate DF affected 18 subjects
(16.6%) and the severe DF affected 5 subjects (4.7%).
Table 2 summarizes the characteristics of the studied population.
There were no significant differences in age and gender between the
groups (p > 0.05). Afro-descendants had a higher risk of DF
(p = 0.008; OR = 1.83; CI 95% = 1.18–2.82).
Table 3 presents the genotypic distribution for MMP2, MMP9 and
MMP20 between DF and DF free groups. Although borderline associa-
tions were observed in MMP2 (rs243865) and MMP9 (rs17576), there
were no statistical associations between genotype and allele in MMP2,
MMP9 and MMP20 distribution and DF (p > 0.05). In the poly-
morphism rs1784418 in MMP20, in a dominant model (AA + AG X
GG), a borderline association was observed (p = 0.06)
Table 4 demonstrates the results of the logistic regression analysis
adjusted by the ethnicity; there was no statistical significance
(p > 0.05).
4. Discussion
The present study showed the immunostaining of the MMP2, MMP9
and MMP20 in the enamel matrix of two mouse strains, one susceptible
and one resistant to DF. It is important to emphasize that the main goal
of this immunohistochemistry step was to evaluate if these MMPs were
expressed during the enamel maturation stage. Immunohistochemistry
has some limitations when the results are expressed in a quantitative
manner (da Silva et al., 2012), quantitative real-time polymerase chain
reaction is more precise to quantify the expression. Thus, only a qua-
litative analysis considering the presence or the absence and localiza-
tion of immunostaining was performed here. Due to the observation of
these immunostainings in the first step of our study, MMP2, MMP9 and
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Environmental Toxicology and Pharmacology 66 (2019) 104–108
Location in the gene Base Change Alteration MAF
promoter C/T down regulation* 0.136
promoter A/G down regulation* 0.363
exon 6 A/G Gln279Arg** 0.455
Intron A/G unknown 0.422
Intron A/G unknown 0.396
MMP20 were considered candidate genes to explore the association of
genetic polymorphisms involved in human susceptibility to DF.
Based on these evidences, we investigated the association between
the genetic polymorphisms rs243865, rs243867, rs17576, rs1784418
and rs1711437 with human DF. The role of genetic susceptibility on DF
is largely unclear. Some studies have been performed on mouse strains
to find the genetic determinants of fluorosis and the association be-
tween fluorosis and genetic polymorphisms. These studies have showed
that genetic variants in some candidate genes could increase or de-
crease the risk of DF among the exposed individuals in endemic areas
(Pramanik and Saha, 2017). However, the present study was the first
one that investigated polymorphisms in MMP2, MMP9 and MMP20
genes in the human population.
There is little evidence of the effect of fluoride on the activity of
ameloblasts, in particular on the expression of proteases; however, the
evidence demonstrates that fluoride down regulates or inhibits the
MMPs during enamel development. Jing et al., (2006) investigated the
effects of overdosed fluoride on the expression of MMP20 in rat incisor.
Immunohistochemical results demonstrated the presence of MMP20 in
ameloblasts. The authors reported that MMP20 was down regulated in
the treated group, in which overdosed fluoride inhibits the secretion of
MMP20 leading to the disturbed balance between MMP20/TIMP2 in rat
enamel, causing the delay of the amelogenin removal and the enamel
demineralization. In our study, both mouse strains received F treat-
ment. Other in vitro studies (Zhang et al., 2006, 2007) have suggested
that fluoride can affect the expression of MMP20, downregulating this
expression by human ameloblast lineage cells. The excessive fluoride
inhibits the expression of MMP20 in rats and mice incisors (Wang et al.,
2016; Zhang et al., 2011), which leads to the delay of amelogenin re-
moval and enamel demineralization. On the other hand, it was de-
monstrated that fluoride did not significantly affect MMP20 expression
during the secretory stage in rat enamel (Suzuki et al., 2014). We ob-
served MMP20 expression in both strains, susceptible and resistant. In
the human population, we studied two intronic variants in MMP20, and
this gene was only borderline associated with DF in the polymorphism
rs1784418, in the recessive and in the multivariate analysis. It is pos-
sible that these polymorphisms in MMP20 are associated with DF in
other populations.
Other evidences of the interaction between fluoride and MMPs have
been observed. Kato et al. (2014) found that fluoride decreased the
activities of pro- and active forms of salivary and purified human
MMPs, including MMP2 and MMP9, in a dose-response manner. Also,
more recently, a significant association was also found between a
genotype of MMP2 and skeletal fluorosis severity in Tibetans and Ka-
zakhs (Pei et al., 2017). In our study we selected and evaluated two
genetic polymorphic variants involved in the down regulation of MMP2
expression. In the genetic polymorphisms rs243865, the genetic variant
associated with the down regulation of MMP2, the A allele, was bor-
derline associated with DF here. The genetic polymorphism rs17576 in
MMP9 studied here is involved in the protein configuration alteration,
in which a substitution of glutamine (Gln) by arginine (Arg) occurs. In
our study, the Gln-Gln genotype was under-represented in DF group and
was also borderline associated (p = 0.08). It is possible that these two
genetic polymorphisms, rs243865 in MMP2 and rs17576 in MMP9
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P.C. Romualdo et al. Environmental Toxicology and Pharmacology 66 (2019) 104–108

Fig. 1. Representative pictures of the 9 weeks

old incisors of animal resistant and susceptible
to DF. Phenotypic characteristics of the mice
incisors of the resistant mouse (A) and sus-
ceptible mouse (B) after 6 weeks of treatment
with fluoride as NaF in the drinking water.
Photomicrographs of the im-
munohistochemical findings using MMP2,
MMP9 and MMP20 antibodies on the incisors,
in a longitudinal section under 40X magnifi-
cation (C–H). C- Immunopositivity for MMP2
in the enamel matrix of resistant mouse. D-
Immunopositivity for MMP2 in the enamel
matrix of susceptible mouse. E-
Immunopositivity for MMP9 in the enamel
matrix of resistant mouse. F- Immunopositivity
for MMP9 in the enamel matrix of susceptible
mouse. G- Immunopositivity for MMP20 in the
enamel matrix of resistant mouse. F-
Immunopositivity for MMP20 in the enamel
matrix of susceptible mouse.

Table 2 Table 3
Demographic data of the studied children. Results of genotype and allele comparisons between dental fluorosis and dental
fluorosis free groups.
Variables DF (n = 108) DF free (n = 373) p-value
Gene and base change rs# DF DF free p-values
Mean Age (SD) 9.53 (2.51) 9.90 (2.85) 0.22
Gender (%) dd/dD/ dd/dD/DD Genotype Allele
Female 54 (50.0) 179 (48.0) 0.74 DD
Male 54 (50.0) 194 (52.0)
Ethnicity (%) MMP2 (C/T) 243,847 7/38/19 31/102/86 0.19 0.51
Caucasian 49 (45.4) 225 (60.3) 0.008 MMP2 (A/G) 243,865 6/14/47 42/108/ 0.17 0.06
Afro-descendants 59 (54.6) 148 (39.7) 207
MMP9 (A/G) 17,576 5/29/33 55/113/ 0.08 0.23
Note: Bold font indicates statistical significance. 189
MMP20 (A/G) 1,711,437 8/27/26 32/87/89 0.89 0.83
MMP20 (A/G) 1,784,418 13/35/22 36/89/99 0.16 0.10
could be associated with DF in a larger sample.
MMPs have been studied in other oral conditions. In periodontal †
Upper case letters denote the more frequent allele in controls; dd is the less
tissues, excessive fluoride intake increases MMP2 (Lutfioglu et al., frequent homozygotyc genotype, DD is the more frequent homozygotyc geno-
2012). Polymorphisms in MMP9 were involved in the susceptibility to type and dD is the heterozygotyc. Chi-square test was used.
chronic periodontitis in Uygur adults in Moyu county of Xinjiang (Ma
et al., 2017). Polymorphisms in MMP2 and MMP3 were associated with (Antunes et al., 2016). Polymorphisms in MMP13 and the tissue in-
periapical lesion formation (Menezes-Silva et al., 2012). MMPs were hibitors of metalloproteinases (TIMP1 and TIMP2) were evaluated in
also associated with caries, in which polymorphisms in MMP9 and DF. Although MMP13 was not associated, TIMP1 was associated with
MMP20 were involved in white spot lesions and early childhood caries

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Table 4 Everett, E.T., Yan, D., Weaver, M., Liu, L., Foroud, T., Martinez-Mier, E.A., 2009.
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TT 0.42 0.68 (0.27-1.73)
1226–1234.
243,847 AA AG 0.30 1.61 (0.65-3.98) Huang, H., Ba, Y., Cui, L., Cheng, X., Zhu, J., Zhang, Y., Yan, P., Zhu, C., Kilfoy, B., Zhang,
GG 0.86 0.91 (0.34-2.40) Y., 2008. COL1A2 gene polymorphisms (Pvu II and Rsa I), serum calcio-tropic hor-
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MMP20 1,711,437 AA AG 0.80 1.07 (0.58-2.00) blastin gene polymorphisms and the susceptibility to dental fluorosis. Zhonghua Liu
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GG 0.21 1.65 (0.75-3.63)
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found in the online version. J.H., 2011. Localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 in human coronal
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