Journal of Photochemistry & Photobiology, B: Biology 237 (2022) 112597

Contents lists available at ScienceDirect

Journal of Photochemistry & Photobiology, B: Biology

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

Laser photobiomodulation does not alter clinical and histological

characteristics of 4-NQO-induced oral carcinomas and leukoplakia in mice
Gabriela Weirich Neculqueo a, Marina Estrázulas b, Karen Cherubini a, Valesca Sander Koth a,
Fernanda Gonçalves Salum a, *
a
School of Health and Life Sciences, Postgraduate Program in Dentistry, Oral Medicine Division, Pontifical Catholic University of Rio Grande do Sul- PUCRS, Porto
Alegre, Rio Grande do Sul, Brazil
b
Medical School, Postgraduate Program in Medicine, Pontifical Catholic University of Rio Grande do Sul- PUCRS, Porto Alegre, Rio Grande do Sul, Brazil

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

Keywords: Objective: This study evaluated the effect of laser photobiomodulation (PBM) on oral leukoplakia and squamous
4-NQO cell carcinomas (OSCC) in a model of oral carcinogenesis.
Oral carcinogenesis Materials and Methods: Forty-one C57Bl/6 female mice were distributed in control group, 4-NQO group, Laser
Mice
group 1.5 J and Laser group 9 J. Oral cancer was induced on the tongue by nitroquinoline oxide (4-NQO), diluted
Photobiomodulation
in the water for 16 weeks. In the 18th and 19th weeks, PBM with a diode laser, 0.028 cm2 spot size, continuous
emission mode, 660 nm wavelength was applied on the tongue of animals for seven sessions. Laser group 1.5 J
received 30 mW power and 1.5 J energy. In the Laser group 9 J, 100 mW power, and 9 J energy were applied. In
the 20th week the animals were euthanized.
Results: All animals exposed to carcinogen developed clinical and histological alterations such as leukoplakia and
OSCC on the tongue. There was no significant difference among Laser groups 1.5 and 9 J and 4-NQO group (not
irradiated) regarding the area of leukoplakia and carcinomas (P > 0.05) or thickness of epithelial tissue and
keratin (P > 0.05). There were also no association between PBM and histologic classification of the lesions (P =
0.87), frequency of OSCC (P = 0.57), grade of tumor differentiation (P = 0.88) or depth of invasion (P = 0.45).
Conclusion: Laser PBM, in both parameters used, does not influence on clinical and histological characteristics of
oral leukoplakia and OSCC.
Clinical Relevance: Results suggest that PBM may be a safe treatment for adverse effects of antineoplastic ther­
apies in patients with leukoplakia and OSCC.

1. Introduction demonstrated in the literature, the Multinational Association of Sup­

portive Care in Cancer (MASCC) and International Society of Oral
Squamous cell carcinoma is the most frequent neoplasm of the oral Oncology (ISOO) indicate the clinical use of PBM for the management of
cavity, with high rates of incidence, morbidity and mortality in the OM. However, due to the limited number of clinical studies proving the
world population over the years [1]. The main treatments modalities for safety of application on tumor cells, they recommend its use with
this neoplasm are still surgical resection, radiotherapy and chemo­ caution and away from these areas [6].
therapy, alone or in association [2]. These therapies have important PBM is a therapeutic modality capable of altering biological activity
adverse effects such as oral mucositis (OM), a debilitating condition, by photon energy [9]. Light energy interacts with the tissue and pro­
which clinically presents as areas of erythema, erosion and ulceration of motes effects analgesic, anti-inflammatory and tissue biomodulation
the mucosa, in different degrees of severity. It causes painful symp­ that can assist in the process of tissue repair [6,10]. In addition, it is
tomatology and dysphagia, and can result in weight loss, malnutrition considered a non-invasive therapy, not present cytotoxic effects or drug
and susceptibility to opportunistic infections [3]. In recent years there interaction, has easy application and good acceptance by patients in
has been an increase in the use of laser photobiomodulation (PBM) for clinical practice [7,11]. Nevertheless, little is known about its action on
the management of OM [4–8]. Based on its positive effects, already dysplastic and neoplastic cells. In vitro studies show controversial

* Corresponding author at: Serviço de Estomatologia – Hospital São Lucas, PUCRS, Av. Ipiranga, 6690 Room 231, CEP: 90610-000 Porto Alegre, RS, Brazil.
E-mail address: [email protected] (F.G. Salum).

https://doi.org/10.1016/j.jphotobiol.2022.112597
Received 4 May 2022; Received in revised form 21 October 2022; Accepted 7 November 2022
Available online 11 November 2022
1011-1344/© 2022 Elsevier B.V. All rights reserved.
G.W. Neculqueo et al. Journal of Photochemistry & Photobiology, B: Biology 237 (2022) 112597

results, while some demonstrate inhibitory effects on tumor cells, others diluted in water with a final concentration of 50 μg/ml. It was admin­
show stimulatory effects on tumor progression [12–14]. However, these istered through water ingestion for 16 weeks. In the 17th week, the
models show little similarity to the pathophysiological conditions of an administration of 4-NQO was interrupted, and only water ad libitum was
organism, in addition to limited cellular interactions [14,15]. available to the animals. The protocol of oral carcinogenesis was per­
So far, only two in vivo studies have analyzed the effect of laser PBM formed according to the methodology described by Ottaviani et al. [16],
in a model of oral carcinogenesis [16,17]. The results obtained were which has been used in the induction of oral squamous cell carcinoma,
opposite, one of the studies showed an increase in the degree of tumor that present a similar appearance to human carcinoma.
differentiation [17], while the other showed a reduction in the incidence Animals (N = 41) were randomized into four groups: control group
of carcinomas in situ and invasive, with the PBM [16]. As they were (N = 9) received propyleneglycol, for 16 weeks in the drinking water
developed in different experimental models, with different chemical (50 μg/ml); 4-NQO group (N = 10) received 4-NQO (50 μg/ml) in the
carcinogenic and irradiation parameters, it is not possible to carry out an drinking water; Laser group 1.5 J (N = 12) received 4-NQO (50 μg/ml)
effective comparison between them [12]. Furthermore, studies suggest and PBM with 1.5 J energy; Laser group 9 J (N = 10) received 4-NQO
that the laser may have a biphasic effect, demonstrating different tissue (50 μg/ml) and PBM with 9 J energy (Fig. 1).
responses with different radiation parameters [18,19].
Therefore, considering the benefits of laser PBM in the management 2.3. Laser Photobiomodulation Therapy
of OM in patients with head and neck cancer and the inconclusive results
about the effects of this therapy in oral epithelial dysplasia or in tumor In the 18th and 19th weeks, Laser groups 1.5 J and 9 J received PBM
cells, this study aimed to evaluate the effect of laser PBM at the red with a diode laser, active medium of indium gallium aluminum phos­
wavelength (660 nm) in a model of 4-NQO-induced oral carcinogenesis phide (InGaAlP) (Photon Lase III – DMC Ltda, São Carlos, SP, Brazil),
in mice. 0.028 cm2 spot size. In the Laser group 1.5 J the continuous emission
mode was used, 660 nm wavelength, 30 mW power, 1.5 J energy per
2. Materials and Methods point, 1071.42 mW/cm2 power density, 53.57 J/cm2 energy density for
50 s, parameters similar to Monteiro et al. [17] and Petrellis et al. [20],
2.1. Animals with some modifications. In the Laser group 9 J, continuous emission
mode was used, 660 nm wavelength, 100 mW power, 9 J energy per
The sample consisted of 48 C57Bl/6 female mice (8-week-old), point, 3571.42 mW/cm2 power density, 321.42 J/ cm2 energy density
weighing 15 to 20 g, obtained from the Center for Experimental Bio­ for 90 s, parameters similar to Ottaviani et al. [16] and Frigo et al. [21],
logical Models (CeMBE/PUCRS, Porto Alegre, RS, Brazil). The study was with modifications (Table 1).
approved by the Ethics Committee on Animals Use (CEUA) of Pontifical The laser PBM occurred at one point on the dorsum of tongue. For
Catholic University of Rio Grande do Sul (PUCRS), Brazil (protocol laser application mice were restrained by the scruff method. The spot tip
number #10277). From 48 mice, one died due to malocclusion, three was placed in contact with the mucosa in an angle as perpendicular as
from stress during laser protocol and three developed cachexia possible, minimizing refraction (Fig. 2). We used a power meter to check
throughout the experiment. Therefore, 41 animals were included in the the output power. The groups performed the treatment for two weeks,
analysis. They were housed in standard micro-isolators (five per cage),
equipped with inlet/outlet air filters, under controlled temperature (23 Table 1
± 1 ◦ C) and humidity (50 ± 5%), and a light- dark cycle of 12 h (lights Irradiation parameters for the different groups.
on at 7 a.m., lights off at 7 p.m.). The cages were filled with autoclaved Group Wavelength Energy Energy Power Power Time
wood chip bedding. The animals received Nuvilab-Cr1 pelleted food (nm) Density (J) (mW) Density (s)
(Nuvilab, Colombo, PR, Brasil) and sterile water ad libitum. They were (J/cm2) (mW/
submitted to a period of acclimatization of approximately 10 days. cm2)
Animals were weighed weekly and before euthanasia [16]. Control 660 53.57 1.5 30 1071.42 50
4-NQO – – – – – –
Laser 660 53.57 1.5 30 1071.42 50
2.2. Oral Carcinogenesis Model and Experimental Protocol 1.5 J
Laser 9 660 321.42 9 100 3571.42 90
Oral carcinogenesis was induced with nitroquinoline oxide (4-NQO – J
Sigma, St. Louis, USA) dissolved in propyleneglycol (4 mg/ml) and

Control group 4-NQO group Laser group 1.5 J Laser group 9 J

(N = 9) (N = 10) (N = 12) (N = 10)

Week 0 Week 0
Administration of Administration of nitroquinoline oxide (4-NQO)
propyleneglycol

Week 16 Week 16
End of administration of End of oral carcinogenesis induction protocol
propyleneglycol

Weeks 18 and 19 Weeks 18 and 19

Laser PBM (7 sessions) Laser PBM (7 sessions)

Week 20
Euthanasia; clinical analysis; tissue collection

Fig. 1. Protocol performed in the experiment.

2
G.W. Neculqueo et al. Journal of Photochemistry & Photobiology, B: Biology 237 (2022) 112597

Fig. 2. Laser irradiation. (A) 4-NQO group without laser activation. (B) Irradiation on the dorsum tongue in 1.5 J laser group. A clamp was used to facilitate opening
the mouth and gauze to avoid irradiation in the eyes.

on alternate days, completing seven sessions. Sham laser irradiation was invasive front was also scored [24].
performed in 4-NQO group. Animals were restrained by the scruff The thickness of the epithelium and keratin was analyzed in all the
method, and the spot tip was placed in contact with the mucosa, how­ samples, however, in animals that developed squamous cell carcinoma,
ever laser appliance was not activated. The control group was irradiated evaluation was performed in adjacent areas, where there was no
with the same parameters of the Laser group 1.5 J. neoplasm. The thickness of the epithelium and keratin were measure by
using the Image J Software (NIH, Bethesda, MD, USA), under 200 X
2.4. Euthanasia, Clinical Analysis and Preparation of Tissue magnification. Three fields of the dorsum of the tongue were captured,
and three measurements were taken in each field. The mean of the nine
After the photobiomodulation protocol, at 20th week, the animals measurements was recorded for each specimen. The histological images
were euthanized with an overdose of deep inhalation anesthesia using of the tongue dorsum were obtained with a microscope Axio Imager A1
4% isoflurane. Immediately after euthanasia, clinical evaluation was coupled to an image capture system Axio Vision Rel. 4.4 Software
performed by a blinded examiner, who evaluated the presence of exo­ Multimedia (Carl Zeiss, Hallbergmoos, Germany).
phytic lesions, compatible with squamous cell carcinomas, and leuko­
plastic lesions on the oral mucosa. The tongues were photographed in a 2.6. Statistical Analysis
standardized way and the images saved in TIFF format for macroscopic
analysis. They were collected and immediately fixed in 10% formalde­ All data were analyzed using SPSS 18.0 software. Kruskal-Wallis non-
hyde, for 24 h. parametric ANOVA test, followed by the pair-by-pair comparison test,
Macroscopic analysis of the leukoplakias and neoplastic lesions of was used to compare tumor and leukoplakia area, depth of invasion,
the tongue was performed by a blind examiner. Previously, the examiner epithelium and keratin thickness, and body weight. Fisher's exact test
was calibrated by analyzing 10 images in duplicate at a one-week in­ was used for the histological classification of lesions, presence of tumors
terval. The measurement of the tumor area as well as of the leukoplakias clinically and histologically detectable, and the grade of tumor differ­
areas was calculated, by using Image J Software (NIH, Bethesda, MD, entiation. P value ≤0.05 was considered as an indicative of significance.
USA).
3. Results
2.5. Histological Analysis
3.1. Clinical Evaluation
The tongue specimens were subjected to routine histological pro­
cessing and embedded in paraffin. Sections of 3 μm thick were obtained The induction of oral carcinogenesis in the 4-NQO group, Laser
and stained with hematoxylin and eosin (H&E). Histological analysis groups 1.5 J and 9 J resulted in whitish plaques on the dorsum, edges
was performed by one calibrated and blinded examiner, using a BX50 and ventral tongue of the animals, compatible with leukoplakia. Exo­
Olympus binocular microscope (Olympus, Tokyo, Japan). Intra- phytic whitish nodular lesions with irregular surface, sessile or pedun­
examiner calibration was performed by reanalysis of 10 slides with an culated, located on the dorsum, edges and ventral tongue, and floor of
interval of seven days between observations. The histological sections the mouth were also found (Fig. 3). These lesions were clinically
were examined in their entirety and were classified into four groups: no compatible with squamous cell carcinoma.
changes; hyperkeratosis and acanthosis; epithelial dysplasia and squa­ In the control group, no alterations were observed in the oral mucosa
mous cell carcinoma [22]. of the animals. In the 4-NQO group, seven animals (70%) developed
In addition, the squamous cell carcinomas were analyzed to verify clinical evidence of neoplasia, in the Laser group 1.5 J, five (41.6%)
the existence of rupture of the basal membrane and were classified ac­ mice had a tumor detected during the examination, and in the Laser
cording to the degree of differentiation in well-differentiated; moder­ group 9 J, six (60%) animals developed those lesions. There was no
ately differentiated and poorly differentiated [16,17,23]. In invasive significant difference in the frequency of clinically detectable tumors
tumors, that is, in which there was rupture of the basal membrane, depth among the carcinogen-treated groups (p = 0.45).
of invasion (or tumor thickness) was measured from the tumor surface to Regarding the tumor and leukoplakia area, there were no significant
the deepest point of invasion by the Image J Software (NIH, Bethesda, differences among the groups in which oral carcinogenesis was induced,
MD, USA), under 200 X magnification. Tumor budding, defined as the regardless of the laser PBM (p > 0.05). 4-NQO group (18.40 mm2 ±
presence of a single cancer cell or small cluster of <5 cancer cells at the 4.34), Laser groups 1.5 J (15.19 mm2 ± 5.84) and 9 J (21.26 mm2 ±

3
G.W. Neculqueo et al. Journal of Photochemistry & Photobiology, B: Biology 237 (2022) 112597

Fig. 3. Representative images of the tongues. Control animal treated with vehicle, without lesions (A). Animals treated with 4-NQO (50 μg/ml) in drinking water
showing leukoplakia on the dorsum of the tongue (B,C). Animals treated with 4-NQO presenting multiple neoplastic exophytic lesions with irregular surface on the
dorsum (D,E) and ventral tongue (F).

6.43) showed similar values regarding those variables (Fig. 4A). among the groups subjected to induction of oral carcinogenesis,
The weight gain of the animals throughout the study was lower in the regardless of PBM therapy (p = 0.80, Fig. 6D). Depth of invasion (or
4-NQO group (2.18 g ± 2.12), Laser groups 1.5 J (0.11 g ± 1.81) and 9 J tumor thickness) was 0.508 mm (± 0.284) in 4-NQO group, 0.340 mm
(1.90 g ± 1.59), compared to control group (4.27 g ± 2.41). Only Laser (± 0.146) in Laser 1.5 J group, and 0.535 mm (± 0.213) in Laser 9 J
group 1.5 J showed a significant difference compared to control group group. There was not significant difference between groups regarding to
(p < 0.05) (Fig. 4B). depth of invasion (p = 0.455). Tumor budding were not detected in the
samples of squamous cell carcinoma.

3.2. Histological Analysis 4. Discussion

There was a statistically significant increase in the thickness of the The present study evaluated the effects of laser PBM on leukoplakia
epithelium and keratin in the dorsum of the tongue in 4-NQO (273.70 and oral squamous cell carcinomas in a 4-NQO-induced carcinogenesis
μm ± 30.60), Laser 1.5 J (275.40 μm ± 83.70) and Laser 9 J (256.90 μm model. Laser with both parameters of energy, 1.5 J and 9 J, did not
± 49.62) groups compared to control (108.00 μm ± 27.85) (p < 0.001). change the frequency of clinically detectable tumors at the end of the
Nevertheless, no significant differences were observed among the experiment. Despite the slight increase in the area of lesions in the Laser
groups subjected to induction of oral carcinogenesis, receiving or not 9 J group, the area of leukoplakia and carcinomas also did not show any
PBM therapy (P = 0.89) (Fig. 4C-G). changes with PBM in neither parameters. Our study is unprecedented in
In the control group, no alterations were observed in the histopath­ performing this clinical analysis, as no in vivo study evaluating the ef­
ological analysis (Fig. 5A). In the groups exposed to oral carcinogen, all fects of PBM on leukoplakia and oral carcinomas has analyzed the area
samples developed some epithelial histological alteration (Fig. 5B-F). In of clinically detectable lesions. The results showed that with low en­
the 4-NQO group, squamous cell carcinoma was detected in eight mice ergies, like those used in the treatment of oral mucositis, and with high
(80%) and epithelial dysplasia in two (20%). In the Laser group 1.5 J, energies, PBM did not change the clinical parameters of oral lesions
carcinomas were observed in seven animals (58.3%), dysplasia in three induced by 4-NQO.
(25%) and hyperkeratosis and acanthosis in two (16.7%). In the Laser Regarding the histopathological analysis, more cases of squamous
group 9 J were also diagnosed seven carcinomas (70%), two dysplasias cell carcinomas were diagnosed compared to the clinical analysis. In the
(20%) and one hyperkeratosis and acanthosis (10%) (Fig. 6A). Fisher's 1.5 J laser group, two more cases were detected and in the 4-NQO and 9
Exact test didn't demonstrated association between the histological J laser groups, one more sample. As in previously published studies, in
classification of lesions and laser PBM in animals subjected to induction this experiment all animals exposed to the carcinogen showed histo­
of oral carcinogenesis (X2(4) = 2.084; p = 0.87) (Fig. 6A). In addition, logical alterations in the tongue, which ranged from hyperkeratosis and
there was no association between the frequency of squamous cell car­ acanthosis, dysplasia to squamous cell carcinoma, with distinct grades of
cinomas and PBM therapy (X2(2) = 1.202; p = 0.57) (Fig. 6B). differentiation [16,22,25]. PBM therapy did not change the histological
According to the Fisher's Exact test, it there was also no association presentation of the lesions or the grade of differentiation of squamous
between the grade of tumor differentiation and PBM therapy (X2(4) = cell carcinomas. In addition, the data showed that the laser did not in­
1824; p = 0.88) (Fig. 6C). Some tumors showed a proliferative growth, fluence in the incidence of tumor invasion among the groups or depth of
without rupture of the basal layer, whereas others showed an invasive invasion.
character. There was no difference regarding this histological aspect

4
G.W. Neculqueo et al. Journal of Photochemistry & Photobiology, B: Biology 237 (2022) 112597

Fig. 4. Effects of 4-NQO-induced oral carcinogenesis and photobiomodulation (PBM). (A) The tumor and leukoplakia area did not differ in animals carcinogen-
treated, receiving or not PBM. (B) Weight gain of C57Bl/6 mice throughout the study (Δ body weight). (C) Epithelium and keratin thickness in 4-NQO, Laser
1.5 J and Laser 9 J groups compared to the control group. (D-G) Representative images showing epithelium and keratin thickness (bidirectional arrow) in control (D),
4-NQO group (E), Laser group 1.5 J (F) and Laser group 9 J (G) (H&E staining). Original magnification 200 X, scale bar (—) 100 μm. ** p < 0.01 compared to
control group.

In contrast, Monteiro et al. [17] observed an increase in the histo­ used irradiation substantially different parameters from those of the
logical grade of squamous cell carcinoma with laser PBM. However, in present study such as infrared wavelength (970 nm), 2.5 W power, 75 J
his study, oral carcinomas were induced in the cheek pouch of golden energy, for 30 s, applied for four consecutive days. This extremely high
Syrian hamsters in an experimental model with 9,10-dimethyl 1,2- power may reduce the effect of photobiomodulation and cause thermal
benzanthracene (DMBA). The laser was applied for 133 s, with energy of alterations, which may explain the reduction of the incidence of the
4 J, for four weeks, on alternate days. This carcinogenic model, induced lesions observed in the study by Ottaviani et al. [16]. Furthermore, these
in hamster cheek pouch, does not represent the environmental in­ parameters could not be used in the management of OM, as they are not
fluences occurred in the oral cavity. Besides, epithelium in this recommended by MASCC and ISOO. Nonetheless laser has biphasic ef­
anatomical region is thinner, compared to the epithelium of the oral fects, being able to demonstrate contrasting responses with different
cavity in humans, which may justify the contrasting results [23,25]. In doses in the same tissue [18,19]. For this reason, in the present study, we
the present study, we opted for the experimental model of induction of opted to use two laser protocols, one with lower energy, similar to that
oral carcinogenesis with 4-NQO in mice, as it presents good results used in clinical practice, and the other with higher energy, which may
already described in the literature [16,22,25,26], added its similarity to have inhibitory effects, without thermal effects.
human carcinomas [22,25]. Futhermore, 4-NQO has advantages over In vitro studies as demonstrated by Gomes Henriques et al. [14]
other chemical carcinogenesis such as DMBA, for being water-soluble, tested two protocols of red laser on tumor cells (SCC25) with a 0.99 J
allowing the administration in the drinking water, causing less stress energy. A stimulatory effect on tumor proliferation and invasion
during the experiment [22,26]. occurred. Our study, using an in vivo model of oral carcinogenesis, with
Ottaviani et al. [16] also found that PBM with laser is a safety pro­ similar irradiation patterns, did not show significant differences in the
cedure in dysplastic and neoplastic lesions. The authors performed an in tumor incidence, invasion and differentiation. The difference among the
vivo study in a 4-NQO-induced model of oral carcinogenesis. However, results can be attributed to the in vitro model, which show little simi­
different from our data, the authors observed a reduction in the larity to the pathophysiological conditions [14,15].
appearance of dysplastic lesions, close to the areas of carcinomas, in This study demonstrated multifocal leukoplastic and neoplastic le­
addition to a lower incidence of in situ and invasive carcinomas. They sions after the induction with 4-NQO in the drinking water (50 μg/ml),

5
G.W. Neculqueo et al. Journal of Photochemistry & Photobiology, B: Biology 237 (2022) 112597

Fig. 5. Representative images of histological aspect of tongues after treatment with 4-NQO and vehicle. (A) Control group without alterations, sample treated with
vehicle. (B) Hyperkeratosis and acanthosis in group laser 1.5 J. (C) Epithelial dysplasia in group laser 9 J. (D) Squamous cell carcinoma with exophytic growth in
laser group 9 J. (E and F) Invasive squamous cell carcinoma in laser group 1.5 J. (H&E staining). Original magnification 100 X (A, D and E) and 200 X (B, C and F).

in the 20th week of the experiment. These lesions developed throughout euthanize the animals at different times, that is, after the PBM protocol
the entire dorsum of the tongue of animals. Furthermore, some of the (as performed) and a few months later. Thus, it would be possible to
animals also developed lesions in the ventral tongue, in the edges of evaluate the effects of PBM on tumor development and on clinical and
tongue and in the mouth floor. Such lesions are compatible with what is histological changes over the time. Another issue to be discussed is that
described in the literature using the same experimental model [16,22]. the carcinomas observed in this study had a leukoplakia and exophytic
Therefore, we decided to assess the thickness of epithelium and keratin clinical appearance. It would also be interesting to observe the effect of
from the dorsum of the tongue of the animals, in fields where there was PBM on lesions with an erythroplakia and endophytic clinical
no squamous cell carcinoma. The data showed an increase in these appearance.
variables in the groups that used the carcinogen, but no differences The laser PBM, in the parameters of the present study, did not show
among them. This indicates that laser PBM, in the presented parameters, significant influence in relation to the area of oral leukoplakia and
does not have the ability to change the thickness of epithelium and carcinomas, or in relation to the histological classification of lesions,
keratin. In vivo studies that evaluated the effects of PBM on oral carci­ tumor incidence, grade of tumor differentiation, thickness of the
nogenesis model did not analyze these parameters until then. On the epithelium and keratin. This implies that laser PBM in these parameters
other hand, a study in a skin carcinogenesis model, which evaluated the is not able to change the clinical and histopathological features of oral
application of LED for 30 min, 642 nm wavelength, 21.6 J/cm2 energy squamous cell carcinomas and leukoplakia in the proposed model. There
density, demonstrated an increase in the thickness of the epithelium, is still a need for further studies, with a larger size sample, a longer
compared to the control group [27]. follow-up period after PBM therapy, and in other oral carcinogenesis
According to the literature, the induction of oral carcinogenesis with models.
4-NQO causes a drastic reduction in the weight of the animals [22]. At
the end of the experiment, the animals in the 4-NQO, laser 1.5 J and Author Contributions
laser 9 J groups showed lower weight gain than the control group.
Nevertheless, only the laser 1.5 J group differed statistically from the Gabriela Weirich Neculqueo: conceptualization, methodology,
control. In the laser 1.5 J group, some animals developed cachexia at the formal analysis and investigation, writing – figures preparation,
end of the experiment. In the 4-NQO and laser 9 J groups, the condition resources
also developed, but the animals were euthanized before the end of the Marina Estrázulas: methodology, formal analysis
experiment and were not included in the data analysis. Some of these Valesca Sander Koth: formal analysis and investigation, writing –
animals developed exophytic tumors of large proportions, causing dif­ figures preparation
ficulty in feeding, associated with cachexia. It is worth noting that the Karen Cherubini: writing - review and editing, supervision
present study presented limitations like the small sample and a short Fernanda Gonçalves Salum: conceptualization, methodology,
time of follow-up after PBM therapy, which may be a factor to consider, writing – figures preparation, writing - review and editing, supervision
as tumor development and progression are time dependent
[22,23,25,28]. To overcome this limitation, it would be possible to

6
G.W. Neculqueo et al. Journal of Photochemistry & Photobiology, B: Biology 237 (2022) 112597

Fig. 6. Effects of 4-NQO-induced oral carcinogenesis and PBM therapy in the histological classification of lesions (A), tumor incidence (B) and grade of tumor
differentiation (C) in each group. Assessment of the frequency of invasive and proliferative carcinomas in the groups (D). ** p < 0.01.

Funding [3] C. Pulito, A. Cristaudo, C. La Porta, S. Zapperi, G. Blandino, A. Morrone, S. Strano,

Oral mucositis: the hidden side of cancer therapy, J. Exp. Clin. Cancer Res. 39
(2020) 1–15, https://doi.org/10.1186/s13046-020-01715-7.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - [4] J.A.E.M. Zecha, J.E. Raber-Durlacher, R.G. Nair, J.B. Epstein, S. Elad, M.
Brasil (CAPES)- finance code 001. R. Hamblin, A. Barasch, C.A. Migliorati, D.M.J. Milstein, M.T. Genot, L. Lansaat,
R. van der Brink, J. Arnabat-Dominguez, L. van der Molen, I. Jacobi, J. van Diessen,
J. de Lange, L.E. Smeele, M.M. Schubert, R.J. Bensadoun, Low-level laser therapy/
photobiomodulation in the management of side effects of chemoradiation therapy
Declaration of Competing Interest
in head and neck cancer: part 2: proposed applications and treatment protocols,
Support Care Cancer 24 (2016) 2793–2805, https://doi.org/10.1007/s00520-016-
The authors declare there is no conflict of interests. 3153-y.
[5] J.A.E.M. Zecha, J.E. Raber-Durlacher, R.G. Nair, J.B. Epstein, S.T. Sonis, S. Elad, M.
R. Hamblin, A. Barasch, C.A. Migliorati, D.M.J. Milstein, M.T. Genot, L. Lansaat,
Data availability R. van der Brink, J. Arnabat-Dominguez, L. van der Molen, I. Jacobi, J. van Diessen,
J. de Lange, L.E. Smeele, M.M. Schubert, R.J. Bensadoun, Low level laser therapy/
No data was used for the research described in the article. photobiomodulation in the management of side effects of chemoradiation therapy
in head and neck cancer: part 1: mechanisms of action, dosimetric, and safety
considerations, Support Care Cancer 24 (2016) 2781–2792, https://doi.org/
Appendix A. Supplementary Data 10.1007/s00520-016-3152-z.
[6] Y. Zadik, P.R. Arany, E.R. Fregnani, P. Bossi, H.S. Antunes, R.J. Bensadoun, L.
A. Gueiros, A. Majorana, R.G. Nair, V. Ranna, W.J.E. Tissing, A. Vaddi, R. Lubart,
Supplementary data to this article can be found online at https://doi. C.A. Migliorati, R.V. Lalla, K.K.F. Cheng, S. Elad, Systematic review of
org/10.1016/j.jphotobiol.2022.112597. photobiomodulation for the management of oral mucositis in cancer patients and
clinical practice guidelines, Support Care Cancer 27 (2019) 3969–3983, https://
doi.org/10.1007/s00520-019-04890-2.
References [7] S.S. Rezk-Allah, H.M.A. Elshafi, R.J. Farid, M.A.E. Hassan, S.A. Alsirafy, Effect of
low-level laser therapy in treatment of chemotherapy induced oral mucositis,
[1] Global Cancer Observatory (n.d.), https://gco.iarc.fr/tomorrow/en (accessed J. Lasers Med. Sci. 10 (2019) 125–130, https://doi.org/10.15171/jlms.2019.20.
March 12, 2022). [8] R. Hanna, S. Dalvi, S. Benedicenti, A. Amaroli, T. Sălăgean, I.D. Pop, D. Todea, I.
[2] A. Chamoli, A.S. Gosavi, U.P. Shirwadkar, K.V. Wangdale, S.K. Behera, N. R. Bordea, Photobiomodulation therapy in oral mucositis and potentially
K. Kurrey, K. Kalia, A. Mandoli, Overview of oral cavity squamous cell carcinoma: malignant oral lesions: a therapy towards the future, Cancers (Basel) 12 (2020)
risk factors, mechanisms, and diagnostics, Oral Oncol. 121 (2021), 105451, 1–14, https://doi.org/10.3390/cancers12071949.
https://doi.org/10.1016/j.oraloncology.2021.105451.

7
G.W. Neculqueo et al. Journal of Photochemistry & Photobiology, B: Biology 237 (2022) 112597

[9] G.C. Louzeiro, K. Cherubini, M.A.Z. de Figueiredo, F.G. Salum, Effect of [19] N.N.F. Lopes, H. Plapler, M.C. Chavantes, R.V. Lalla, E.M. Yoshimura, M.T.S. Alves,
photobiomodulation on salivary flow and composition, xerostomia and quality of Cyclooxygenase-2 and vascular endothelial growth factor expression in 5-fluoro­
life of patients during head and neck radiotherapy in short term follow-up: a uracil-induced oral mucositis in hamsters: evaluation of two low-intensity laser
randomized controlled clinical trial, J. Photochem. Photobiol. B Biol. 209 (2020), protocols, Support Care Cancer 17 (2009) 1409–1415, https://doi.org/10.1007/
https://doi.org/10.1016/j.jphotobiol.2020.111933. s00520-009-0603-9.
[10] K.A.M. Kalhori, F. Vahdatinia, M.R. Jamalpour, P. Vescovi, C. Fornaini, E. Merigo, [20] M.C. Petrellis, L. Frigo, R.L. Marcos, R.C. Pallotta, M.H.C. de Carvalho, M.
R. Fekrazad, Photobiomodulation in oral medicine, photobiomodulation, N. Muscará, D.A. Maria, R.Á.B. Lopes-Martins, Laser photobiomodulation of pro-
photomedicine, Laser Surg. 37 (2019) 837–861, https://doi.org/10.1089/ inflammatory mediators on Walker tumor 256 induced rats, J. Photochem.
photob.2019.4706. Photobiol. B Biol. 177 (2017) 69–75, https://doi.org/10.1016/j.
[11] M.D. Martins, F.M. Silveira, L.P. Webber, V.P. Wagner, M.A.T. Martins, C. jphotobiol.2017.09.011.
H. Squarize, R.M. Castilho, The impact of photobiomodulation therapy on the [21] L. Frigo, J.M. Cordeiro, G.M. Favero, D.A. Maria, E.C.P. Leal-Junior, J. Joensen, J.
biology and behavior of head and neck squamous cell carcinomas cell lines, M. Bjordal, D.C. Roxo, R.L. Marcos, R.A.B. Lopes-Martins, High doses of laser
J. Photochem. Photobiol. B Biol. 209 (2020), https://doi.org/10.1016/j. phototherapy can increase proliferation in melanoma stromal connective tissue,
jphotobiol.2020.111924. Lasers Med. Sci. 33 (2018) 1215–1223, https://doi.org/10.1007/s10103-018-
[12] F.M. Silveira, M.D.P. Paglioni, M.M. Marques, A.R. Santos-Silva, C.A. Migliorati, 2461-5.
P. Arany, M.D. Martins, Examining tumor modulating effects of [22] X.H. Tang, B. Knudsen, D. Bemis, S. Tickoo, L.J. Gudas, Oral cavity and esophageal
photobiomodulation therapy on head and neck squamous cell carcinomas, carcinogenesis modeled in carcinogen-treated mice, Clin. Cancer Res. 10 (2004)
Photochem. Photobiol. Sci. 18 (2019) 1621–1637, https://doi.org/10.1039/ 301–313, https://doi.org/10.1158/1078-0432.CCR-0999-3.
c9pp00120d. [23] B.L. Hawkins, B.W. Heniford, D.M. Ackermann, M. Leonberger, S.A. Martinez, F.
[13] J.L. da Silva, A.F.S. Silva-de-Oliveira, R.A.C. Andraus, L.P. Maia, Effects of low J. Hendler, 4NQO carcinogenesis: a mouse model of oral cavity squamous cell
level laser therapy in cancer cells—a systematic review of the literature, Lasers carcinoma, Head Neck 16 (1994) 424–432, https://doi.org/10.1002/
Med. Sci. 35 (2020) 523–529, https://doi.org/10.1007/s10103-019-02824-2. hed.2880160506.
[14] Á.C. Gomes Henriques, F. Ginani, R.M. Oliveira, T.S.L. Keesen, C.A. Galvão [24] R.D. Almangush, I.O. Coletta, C. Bello, H. Bitu, L.K. Keski-Säntti, J.H. Mäkinen,
Barboza, H.A. Oliveira Rocha, J.F.L. De Castro, R. Della Coletta, R. De Almeida M. Kauppila, J. Pukkila, J. Hagström, S. Laranne, Y. Tommola, V.M. Soini,
Freitas, Low-level laser therapy promotes proliferation and invasion of oral P. Kosma, L.P. Koivunen, P. Kowalski, R. Nieminen, I. Grénman, T. Leivo, Salo, a
squamous cell carcinoma cells, Lasers Med. Sci. 29 (2014) 1385–1395, https://doi. simple novel prognostic model for early stage oral tongue cancer, Int. J. Oral
org/10.1007/s10103-014-1535-2. Maxillofac. Surg. 44 (2015) 143–150, https://doi.org/10.1016/j.
[15] J.P. Gillet, S. Varma, M.M. Gottesman, The clinical relevance of cancer cell lines, ijom.2014.10.004.
J. Natl. Cancer Inst. 105 (2013) 452–458, https://doi.org/10.1093/jnci/djt007. [25] J.M. Nauta, J.L.N. Roodenburg, P.G.J. Nikkels, M.J.H. Witjes, A. Vermey,
[16] G. Ottaviani, V. Martinelli, K. Rupel, N. Caronni, A. Naseem, L. Zandonà, Comparison of epithelial dysplasia-the 4NQO rat palate model and human oral
G. Perinetti, M. Gobbo, R. Di Lenarda, R. Bussani, F. Benvenuti, M. Giacca, mucosa, Int. J. Oral Maxillofac. Surg. 24 (1995) 53–58, https://doi.org/10.1016/
M. Biasotto, S. Zacchigna, Laser therapy inhibits tumor growth in mice by S0901-5027(05)80857-4.
promoting immune surveillance and vessel normalization, EBioMedicine. 11 [26] M.C.A. Rivera, 4NQO carcinogenesis: a model of oral squamous cell carcinoma, Int.
(2016) 165–172, https://doi.org/10.1016/j.ebiom.2016.07.028. J. Morphol. 30 (2012) 309–314, https://doi.org/10.4067/s0717-
[17] J.S. Monteiro, A.L.B. Pinheiro, S.C.P.S. De Oliveira, G.T.S. Aciole, J.A.C. Sousa, M. 95022012000100055.
C.T. Cangussú, J.N. Dos Santos, Influence of laser phototherapy (λ660nm) on the [27] H. Goo, S. Mo, H.J. Park, M.Y. Lee, J.-C. Ahn, Treatment with LEDs at a wavelength
outcome of oral chemical carcinogenesis on the hamster cheek pouch model: of 642 nm enhances skin tumor proliferation in a mouse model, Biomed. Opt.
histological study, Photomed. Laser Surg. 29 (2011) 741–745, https://doi.org/ Express. 12 (2021) 5583, https://doi.org/10.1364/boe.427205.
10.1089/pho.2010.2896. [28] N.E. Steidler, P.C. Reade, Experimental induction of oral squamous cell carcinomas
[18] M.R. Hamblin, Y.Y. Huang, S.K. Sharma, J. Carroll, Biphasic dose response in low in mice with 4-nitroquinolone-1-oxide, Oral Surg. Oral Med. Oral Pathol. 57 (1984)
level light therapy - an update, Dose-Response. 9 (2011) 602–618, https://doi.org/ 524–531, https://doi.org/10.1016/0030-4220(84)90312-8.
10.2203/dose-response.11-009.Hamblin.