UNIVERSIDADE ESTADUAL PAULISTA

“JÚLIO DE MESQUITA FILHO”

Instituto de Ciência e Tecnologia

Campus de São José dos Campos

ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2023.e3790

Braz Dent Sci 2023 Apr/Jun;26 (2): e3790

Estrogen deﬁciency inﬂuences TNF-α and IL-1β gene expression in

the odontogenic region of dental hypofunctional condition

A deﬁciência de estrógeno inﬂuencia a expressão gênica de TNF-α e IL-1β na região odontogênica de dentes em hipofunção

Igor Domingos dos ANJOS

, Vinícius Otávio NOGUEIRA

, Martinelle Ferreira da Rocha TARANTO

,

Lucas Alexandre RAMAZZOTTO

, Paulo NELSON-FILHO

, Erika Calvano KÜCHLER

4,5

,

Maria Angélica Hueb de MENEZES-OLIVEIRA

, César Penazzo LEPRI

, Flares BARATTO-FILHO

5,6

,

Isabela Ribeiro MADALENA

1,4,6



1 - Centro Universitário Presidente Tancredo de Almeida Neves, Faculdade de Odontologia. São João del Rei, MG, Brazil.

2 - Universidade Federal de São Carlos, Escola de Biotecnologia. São Carlos, SP, Brazil

3 - Universidade de São Paulo, Faculdade de Odontologia de Ribeirão Preto, Departamento de Odontopediatria. Ribeirão Preto, SP, Brazil

4 - Universidade de Uberaba, Departamento de Biomateriais. Uberaba, MG, Brazil.

5 - Universidade Tuiuti do Paraná, Faculdade de Odontologia. Curitiba, PR, Brazil.

6 - Universidade da Região de Joinville, Departamento de Odontologia. Joinville, SC, Brazil.

How to cite: Anjos ID, Nogueira VO, Taranto MFR, Ramazzotto LA, Nelson-Filho P, Küchler EC, et al. Estrogen deciency inuences

TNF-α and IL-1β gene expression in the odontogenic region of dental hypofunctional condition. Braz Dent Sci. 2023;26(2):e3790.

https://doi.org/10.4322/bds.2023.e3790

ABSTRACT

Objective: Scientic evidence suggests that estrogen deciency and genetic factors have an inuence on the development of the

stomatognathic system. This study aimed to evaluate the inuence of estrogen deciency on the gene expression of TNF-α, IL-1β,

IL-6 and IL-10 during dental development in a murine model. Material and Methods: Wistar Hannover rats were divided into two

groups according to the intervention received: Hypoestrogenism Group - ovariectomy surgery and Control Group - ctitious surgery.

To evaluate the dental development, the lower incisor was chosen. The mandibular incisor hypofunction model was performed

by incisal adjustment. The homologous incisor exerted a hyperfunction. The animals were evaluated throughout the pubertal

period. After euthanasia, the hemimandibles were removed to evaluate the gene expression of the TNF-α, IL-1β, IL-6 and IL-10 in

the odontogenic region of the incisors through real time PCR. Kruskal-Wallis test and Dunn’s posttest were performed. The level

of signicance was 5%. Results: There were statistically signicant differences of TNF-α and IL-1β gene expression between the

hypoestrogenism and control groups under hypofunction condition (p=0.0084, p=0.0072, respectively). Conclusion: Estrogen

deciency inuences TNF-α and IL-1β gene expression in the odontogenic region of the hypofunctional teeth.

KEYWORDS

Osteogenesis; Estrogen; Proinammatory cytokines; Gene Expression; Genes.

RESUMO

Objetivo: Evidências cientícas sugerem que a deciência de estrógeno e fatores genéticos inuenciam o desenvolvimento

do sistema estomatognático. Este estudo teve como objetivo avaliar a inuência da deciência de estrógeno na expressão

gênica de TNF-α, IL-1β, IL-6 e IL-10 durante o desenvolvimento dentário em modelo murino. Material e Métodos: Ratas

Wistar Hannover foram divididas em dois grupos de acordo com a intervenção recebida: Grupo Hipoestrogenismo - cirurgia

de ovariectomia e Grupo Controle - cirurgia ctícia. Para avaliar o desenvolvimento dentário, o incisivo inferior foi escolhido.

O modelo de hipofunção dos incisivos inferiores foi realizado por ajuste incisal. O incisivo homólogo exercia hiperfunção

dentária. Os animais foram avaliados durante todo o período puberal. Após a eutanásia, as hemimandíbulas foram removidas

para avaliar a expressão gênica do TNF-α, IL-1β, IL-6 e IL-10 na região odontogênica dos incisivos por meio de PCR em tempo

real. Foi realizado o teste de Kruskal-Wallis e o pós-teste de Dunn. O nível de signicância foi de 5%. Resultados: Houve

diferenças estatisticamente signicativas na expressão gênica de TNF-α e IL-1β entre os grupos hipoestrogenismo e controle

sob condição de hipofunção dentária (p=0,0084, p=0,0072, respectivamente). Conclusão: A deciência de estrógeno

inuencia a expressão gênica de TNF-α e IL-1β na região odontogênica de dentes hipofuncionais.

PALAVRAS-CHAVE

Osteogênese; Estrogênio; Citocinas pró-inamatórias; Expressão gênica; Genes.

Braz Dent Sci 2023 Apr/Jun;26 (2): e3790

Anjos ID et al.

Estrogen deficiency influences TNF-α and IL-1β gene expression in the odontogenic region of dental hypofunctional condition

Anjos ID et al.

Estrogen deﬁciency inﬂuences TNF-α and IL-1β gene

expression in the odontogenic region of dental hypofunctional

condition

INTRODUCTION

Estrogen is a steroid hormone present

and active throughout an individual’s life [1].

Although it is primarily responsible for female

characteristics, estrogen also plays an important

role in the neuroendocrine, vascular, skeletal,

and immune systems of both sexes [1-3]. Recent

research has attributed great value to knowledge

about the molecular mechanisms that estrogen

and/or its deciency could cause in the human

body [4-10]. Estrogen deficiency is observed

in children through congenital conditions

associated with chromosomal, gonadal, or atypical

phenotype sexual development [11]. Thus, it is

important to know the implications of estrogen

and its deficiency in the development of the

stomatognathic system. Evidence demonstrates

gene overexpression of estrogen receptors (ERβ)

in cells involved odontogenesis and tooth eruption

process in estrogen deficiency [10]. Estrogen

deciency is capable of causing bone formation

impaired by the action of tumor necrosis factor - α

(TNF-α) on mesenchymal cells [12]. TNF-α can

cause an increase in the expression of interleukin

1β (IL-1β) and interleukin 6 (IL-6) [13,14].

Evidence also suggests that IL-6 is the main

cytokine expressed in estrogen deciency [15].

However, interleukin 10 (IL-10) could also be

shown to have an altered expression capable

of inhibiting the differentiation of Th17 cells,

generating the regression of osteoclastogenesis

in the murine model [16].

Dental development is an important

event for the harmonious development of the

stomatognathic system and, consequently,

homeostasis of general health [17,18]. However,

scientic evidence is still needed to understand

the entire physiological process of dental

development, given to their vulnerability to

local, systemic, environmental, and genetic

factors [19-21]. For more than two decades, it

was estimated that over 300 genes are expressed

during dental development. Many of these genes

actively participate in physiological processes [22].

The expression of TNF-α in tooth germs is

described as active in the cytodifferentiation of the

odontogenic epithelium [23,24]; IL-1β stimulates

gene transcription of the odontogenic protein

associated with ameloblast development [25];

Although IL-6 has been widely associated with

pulpal inflammation events [26], it has also

been described as influential in odontogenic

tumors [27], as well as IL-10 [28], suggesting

participation in the stages of odontogenesis.

It is essential that evidence of the interaction

of estrogen deciency, genetics factors and dental

development be elucidated. The physiological

and pathological knowledge of the population

may result in preventive and health promotion

strategies. Thus, the present study aimed to

evaluate the inuence of estrogen deciency on

the gene expression of TNF-α, IL-1β, IL-6, and

IL-10 during dental development in a murine

model.

MATERIAL AND METHODS

Ethical aspects

This research was performed and reported

according to the ARRIVE guidelines [29].

The Ethical Committee in Animal Experimentation

from the School of Dentistry of Ribeirão Preto,

University of São Paulo, Brazil, approved this

study (#2018.40.58.3).

Experimental design

Specimens from the study by Madalena et al. [10],

sectioned hemimandibles, decient or not of estrogen,

Hypoestrogenism Group (n=8) and Control Group

(n=9) respectively, were submitted to the evaluate

the gene expression of TNF-α, IL-1β, IL-6 and IL-10

in the odontogenic region using the technique

real-time quantitative polymerase chain reaction

(RT-qPCR). Conditions of hypofunction (n=4) and

dental hyperfunction (n=4) were performed in the

animals belonging to the Hypoestrogenism Group as

well as, in the Control Group, hypofunction (n=5)

and dental hyperfunction (n=4). The experimental

design can be observed in Figure 1.

The animals came from the Central Bioterium

of the University of São Paulo – Ribeirão Preto

Campus. The animals were requested with

21 days of post-uterine life, which corresponds

to the pre-pubertal period. Animals that failed

in the surgical procedures, that presented dental

fractures during the evaluation of the eruption

rate and that died before the nal evaluation were

excluded from the study. Thus, the animals were

randomly coded and subsequently proceeded to

the randomization of groups and subgroups.

The animals were placed in the Bioterium II at

School of Dentistry of Ribeirão Preto, University of

São Paulo, in a temperature-controlled environment

Braz Dent Sci 2023 Apr/Jun;26 (2): e3790

Anjos ID et al.

Estrogen deficiency influences TNF-α and IL-1β gene expression in the odontogenic region of dental hypofunctional condition

Anjos ID et al.

Estrogen deﬁciency inﬂuences TNF-α and IL-1β gene

expression in the odontogenic region of dental hypofunctional

condition

with a 12-hour light-dark cycle, with free demand

for food (Labina Purina

/Agribrands do Brasil

LTDA, Paulínia, BR) and ltered water.

Estrogen deciency model

To create estrogen deficiency a bilateral

surgical excision of the ovaries (ovariectomy) was

performed in the hypoestrogenism group. While the

control group was submitted to ctitious surgery,

in which the ovaries were moved and returned

to their initial position, as previously described

in Omori et al. [6] and Madalena et al. [10].

Surgical procedures were performed under general

anesthesia, intramuscularly. The drugs used were

10% Ketamine Hydrochloride (Cetamin

10%), at a

dosage of 55mg/kg and 2% Xylazine Hydrochloride

(Xilasyn

), at a dosage of 10mg/kg. After the

surgical procedure, antibiotic, anti-inammatory

and analgesic medication was administered.

The drugs used were Benzylpenicillin Benzathine

(Pentabiotic

) at a dosage of 24,000UI/kg;

Flunixine (Aplonal

1%) at a dosage of 1mg/kg,

both intramuscularly, and Tramadol (Cronidor

2%), at a dosage of 1mg/kg, subcutaneously.

Tramadol is also administered again 24 hours after

the surgical procedure.

The success of the surgical procedure

was confirmed by the animals’ survival,

gradual increase in body weight during the

experimentation period and by the uterine

atrophy after euthanasia in the experimental

group [6,9,10]. The decrease in endogenous

estrogen release, caused by ovariectomy, provides

signicant differences in the body weight and

uterine weight [30].

Dental hypofunction and hyperfunction

conditions

To evaluate the odontogenic region, the

lower incisors were submitted to conditions

of dental hypofunction and hyperfunction.

The dental hypofunction condition was

performed by incisal adjustment, at the level of

the gingival papilla, specically the mandibular

right incisor [9,10,31,32]. Consequently, the

homologous mandibular incisor exerted a

hyperfunction condition [9,10,31,32]. Mondays,

Wednesdays and Fridays were dened, allowing

an interval of 48 and 72 hours between incisal

edge adjustments. Adjustments were performed

for 21 consecutive days (during the entire

pubertal period). The incisal adjustment was

performed with a 7011 double-sided diamond

disc (KG Sorensen

, Cotia, BR) with a thickness

of 0.18 mm.

Euthanasia and preparation of specimens

Euthanasia was performed following the

guidelines of the National Council for the

Control of Animal Experimentation – CONCEA,

through anesthetic overdose with Ketamine

Hydrochloride (300mg/kg of weight) and

Xylazine Hydrochloride (30mg/kg of weight)

associated with decapitation. Therefore, the

hemimandibles were removed, dissected, and

sectioned to isolate the odontogenic region of

the dental organ for gene expression analysis as

illustrated in previous studies [9,10].

Analysis of TNF-α, IL-1β, IL-6 e IL-10 in

odontogenic region – RT-qPCR

The specimens were kept in RNAlater (Life

Technologies Corporation - Carlsbad

, Canada,

USA) and frozen at -80ºC until the day of

processing. The mirVana ™ miRNA Isolation kit

(Thermo Fischer Scientic, Carlsbad, USA) was

used to extract total RNA. Complementary DNA

(cDNA) was synthesized by reverse-transcription

with a Hight Capacity Kit (Applied Biosystems,

Foster City, CA, USA). RT-qPCR was carried out

Figure 1 - Experimental design

Braz Dent Sci 2023 Apr/Jun;26 (2): e3790

Anjos ID et al.

Estrogen deficiency influences TNF-α and IL-1β gene expression in the odontogenic region of dental hypofunctional condition

Anjos ID et al.

Estrogen deﬁciency inﬂuences TNF-α and IL-1β gene

expression in the odontogenic region of dental hypofunctional

condition

on a StepOnePlus

sequence detection system

(Applied Biosystems™, Foster City, CA, USA) using

TaqMan

primers and probes (Thermo Fisher

Scientic, MA, USA) for TNF-α (Rn9999917-m1),

IL-1β (Rn00580432-m1), IL-6 (Rn01410330-m1)

e IL-10 (Rn00563409-m1). GAPDH

(Rn01462661-g1) and ACTB (Rn01412977-g1)

were used as endogenous controls. The relative

levels of mRNA expression were determined by

the 2

-∆∆

Cycle Threshold (2

-∆∆CT

) method [16].

Both, GAPDH and ACTB, genes were used for

sample normalization to calculate the relative

quantification. The mean of both genes was

used. All procedures were performed following

the respective manufacturer’s instructions and

according to established protocols.

Statistical analysis

The data were evaluated using the GraphPad

Prism 7.04 software (GraphPad Software

, La

Jolla, USA). The gene expression of the TNF-α,

IL-1β, IL-6 e IL-10 were expressed as mean ±

standard deviation (SD). Kruskal-Wallis test and

Dunn’s posttest were performed. The level of

signicance was 5%. A post hoc power estimation

was performed in Clincalc.com

RESULTS

Body weight gain was higher in the group

submitted to hypoestrogenism when compared

to the control group (

p=0.002

). Uterine atrophy

was also noted in the group submitted to

hypoestrogenism when compared to the control

group (

p≤ 0.0001

Table I shows the comparison between

the hypoestrogenism and control groups under

the conditions of dental hypofunction and

hyperfunction. There were statistically signicant

differences of TNF-α and IL-1β gene expression

between the hypoestrogenism and control

groups under condition of dental hypofunction

(

p=0.0084

p=0.0072

, respectively). Power

calculations indicated that we had a power

ranging from 6% to 99%.

DISCUSSION

Gaps in genes involved in dental development

still exist. In parallel, it is important to mention

that the expression of ERβ described in cells

that participate in dental development implies

research that complements the action of estrogen

and its related genes. Thus, the present study

aimed to evaluate the influence of estrogen

deciency on the gene expression of TNF-α, IL-1β,

IL-6, and IL-10 during dental development in

a murine model. Our results demonstrate that

in estrogen deciency and dental hypofunction

conditions, there is a signicant increase in TNF-α

while a signicant decrease in IL-1β is also noted.

Studies in murine models are classic when

the objective is to elucidate local, systemic,

environmental, and genetic influences on

Table I - Gene expression of TNF-α, IL-1β, IL-6 and IL-10 in the odontogenic region in dental organ of the lower incisors of both groups, in teeth

with occlusal hypofunction and hyperfunction

Groups

Hypoestrogenism Control

p-value

Hypofunction

tooth

Hyperfunction

tooth

Hypofunction

tooth

Hyperfunction

tooth

TNF-α

Mean (SD) 2.87 (0.87) 1.97 (0.81) 0.90 (0.23) 1.22 (0.36)

0.0072*

Min.-Max. 2.14 – 4.14 1.42 – 3.18 0.55 – 1.22 0.74 – 1.52

IL-1β

Mean (SD) 0.72 (0.45) 0.35 (0.15) 1.27 (0.33) 0.80 (0.28)

0.0084*

Min.-Max. 0.39 – 1.38 0.14 - 0.51 0.86 – 1.77 0.51 – 1.16

IL-6

Mean (SD) 1.82 (0.67) 0.88 (0.27) 0.93 (0.20) 1.28 (0.82)

0.092

Min.-Max. 1.17 – 2.45 0.56 – 1.22 0.69 – 1.14 0.62 – 2.41

IL-10

Mean (SD) 2.01 (1.17) 0.63 (0.26) 0.83 (0.39) 1.78 (1.18)

0.104

Min.-Max. 0.78 – 3.44 0.37 – 0.99 0.38 – 1.31 0.45 – 2.81

*Statistically signiﬁcant diﬀerence.

Braz Dent Sci 2023 Apr/Jun;26 (2): e3790

Anjos ID et al.

Estrogen deficiency influences TNF-α and IL-1β gene expression in the odontogenic region of dental hypofunctional condition

Anjos ID et al.

Estrogen deﬁciency inﬂuences TNF-α and IL-1β gene

expression in the odontogenic region of dental hypofunctional

condition

the development and process of tooth

eruption [9,10,31-33]. It is noteworthy that

the murine model has continuous development

and eruption of incisors, which results in a

broad regenerative capacity of the odontogenic

region. Furthermore, it is suggested that there is

an important applicability of these studies with

human samples due to the similarity of dental

development stages [9,10,31-33]. Several authors

have also reported that shortening or adjustment

of the incisal edge of the incisor (hypofunctional

condition) can lead to a marked increase in

eruption rate and clearly replicate changes

associated with tooth eruption rate [31,34,35].

It is hypothesized that tooth development may

also change under conditions of hypofunctionality

and hyperfunctionality.

Estrogen is a steroid hormone that has been

extensively studied today [4-10]. In addition to

its physiological importance to many vital tissues

and organs and the pubertal development of

girls and boys [2], estrogenic effects have been

attributed to harmful effects from exposure

to synthetic compounds widely distributed in

the environment. Recent scientific evidence

points to the presence of the main estrogen

receptors, ERα and ERβ, encoded by the

ESR1

and

ESR2

genes, respectively, in the dental organ

of teeth in continuous growth in the estrogen-

deficient murine model, influencing patterns

of development by the supposed action in

mesenchymal cells [8,9].

Some inflammatory cytokines, especially

TNF-α, are increased when estrogen is decient.

It is estimated that TNF-α can inhibit the osteogenic

differentiation of mesenchymal stem cells [12].

Our results corroborate the increase in TNF-α

expression in estrogen deciency. Furthermore, it

is possible to suggest that an inammatory process

occurs with the potential to disrupt odontogenesis

and, consequently, the rate of tooth eruption, as

described by Madalena et al. [10]. In contrast, the

increase in TNF-α expression was not sufcient

to induce IL-6 expression in the continuously

growing tooth organ. It is therefore suggested

that the overexpression of ERβ in the odontogenic

region of incisors of animals subjected to estrogen

deciency [10] has acted as a protective factor

and contributed to the reduction of IL-6 and

IL-1β expression levels in lipopolysaccharide

(LPS)-induced PC-3 cells [36]. LPS is a common

inducer of inammation; exposure leads to the

activation of several components involved in

chronic inammation processes, such as altered

levels of cytokines [36]. It is suggested the need

to complement the results by showing other

cytokines that could be involved in the process

of odontogenesis and even tooth eruption.

It is true to say that the vast majority of

studies and scientic evidence involving gene

expression in teeth during development are

limited to animal models, especially in the

murine model [6,8-10,37,38]. The murine

model allows an interesting reproducibility

of human development because more than

90% of its genome can be divided into regions

corresponding to that of humans. However, such

premises do not exclude the need for further

studies related to the expression of cytokines and

TNF-α in human tooth development.

In summary, this study showed that estrogen

deciency interfered with the gene expression

of the interleukins TNF-α and IL-1β. The lack

of estrogen affect the expression of TNF-α. It is

suggested that odontogenesis undergoes a delay

in its process; moreover, the lack of estrogen

negatively affected the expression of IL-1β,

suggesting that the overexpression of estrogen

receptors ERβ acted as a potentially protective

factor against the inammatory process.

CONCLUSION

Estrogen deciency inuences TNF-α and

IL-1β gene expression in the odontogenic region

in dental hypofunctional condition.

Acknowledgements

We also thank to São Paulo Research

Foundation - FAPESP and the Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior –

CAPES/Brasil.

Author’s Contributions

IDA: Writing-original draft preparation.

VON: Writing-original draft preparation.

MFRT: Writing-original draft preparation.

LAR: Methodology and investigation. PNF:

Conceptualization. ECK: Conceptualization

and funding acquisition. MAHMO: Writing –

review and editing. CPL: Writing—review and

editing. FBF: Writing – review and editing. IRM:

Braz Dent Sci 2023 Apr/Jun;26 (2): e3790

Anjos ID et al.

Estrogen deficiency influences TNF-α and IL-1β gene expression in the odontogenic region of dental hypofunctional condition

Anjos ID et al.

Estrogen deﬁciency inﬂuences TNF-α and IL-1β gene

expression in the odontogenic region of dental hypofunctional

condition

Methodology and investigation and funding

acquisition.

Conict of Interest

The authors declare that they have no

conict of interest.

Funding

This research was funded by the São

Paulo Research Foundation - FAPESP (process

#2015/06866-5 and #2016/08149-1) and Coordenação

de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES-Brasil) – PDPG-POSDOC/Bolsa - CAPES

nº 88887.755620/2022-00.

Regulatory Statement

This study was conducted in accordance

with all the provisions of the local human

subjects oversight committee guidelines and

policies of: The Ethical Committee in Animal

Experimentation from the School of Dentistry of

Ribeirão Preto, University of São Paulo, Brazil.

The approval code for this study is: 2018.40.58.3.

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Date submitted: 2022 Jan 26

Accept submission: 2023 Mar 04

Isabela Ribeiro Madalena

(Corresponding address)

Centro Universitário Presidente Tancredo de Almeida Neves, Faculdade de

Odontologia. São João del Rei, MG, Brazil.

Email: isabelarmadalena@hotmail.com

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