Vol.5, No.9, 972-978 (2013) Natural Science
http://dx.doi.org/10.4236/ns.2013.59118
Glycine supplementation reduces the severity
of chemotherapy-induced oral mucositis
in hamsters
Odara Maria de Sousa Sá1, Nilza Nelly Fontana Lopes2, Maria Teresa Seixas Alves3,
Rajesh V. Lalla4, Maria Luiza Vilela Oliva5, Eliana Maria Monteiro Caran6
1Department of Pediatrics, Federal University of São Paulo, São Paulo, Brazil;
2Former Head Division of Dentistry, Pediatric Oncology Institute, São Paulo, Brazil;
3Department of Pathology, Federal University of São Paulo, São Paulo, Brazil;
4Section of Oral Medicine, University of Connecticut Health Center, Farmington, USA;
5Department of Biochemistry, Federal University of São Paulo, São Paulo, Brazil;
6Department of Pediatrics, IOP/GRAACC Medical School of Federal University of São Paulo, São Paulo, Brazil;
Received 29 June 2013; revised 29 July 2013; accepted 7 August 2013
Copyright © 2013 Odara Maria de Sousa Sá et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Objective: Oral mucositis (OM) is a devastating
toxicity associated with cytotoxic cancer ther-
apy. The OM pathogenesis and the complex in-
teractions occur in response to tissue insult.
Application of this evolving model has aided in
the development of mechanistically based the-
rapies for the prevention and treatment of mu-
cositis. The present study was to assess the
effects of glycine supplementation on chemo-
therapy-induced oral mucositis. Methods: In a
hamster cheek pouch model of chemotherapy-
induced oral mucositis, one group of 20 animals
received systemic glycine supplementation for 7
days, while another similar control group did
not. Clinical mucositis severity and neutrophil
infiltrate (on histology) were assessed by blind-
ed examiners. Free r adic al pr oduction w as mea-
sured as malondialdehyde (MDA) le vels. Results:
As compared to control animals, glycine-treated
animals demonstrated a highly significant re-
duction in clinical severity of oral mucositis,
neutrophil infiltrate, and MDA levels (p < 0.001
for all). Conclusions: Glycine supplementation
reduces the severity of chemotherapy-induced
oral mucositis in an animal model. This effect is
at least partly mediated through inhibition of the
inflammatory response and reduced production
of damaging free radicals.
Keywords: Glycine; Chemotherapy; Oral Mucositis;
Neutrophils; Malondialdehyde; Inflammatory
1. INTRODUCTION
Oral mucositis (OM) presents as erythematous and ul-
cerative lesions of the oral mucosa, secondary to chemo-
therapy and/or radiation therapy for cancer [1-3]. Clini-
cal manifestations of OM include intense pain, interfere-
ence with ingestion of food and drink, and impaired
communication. Moreover, infection associated with oral
mucosal lesions can progress to life threatening sepsis
during periods of intense immunosuppression [4]. OM
impacts negatively on patients’ survival and quality of
life, and is associated with longer hospitalizations and
higher costs [5,6]. Perhaps most importantly, dose-re-
ductions in cancer therapy due to mucositis can adversely
affect the outcomes of cancer treatment. However, treat-
ment options for OM are very limited and current man-
agement strategies are largely palliative [7].
OM is an inflammatory response of the oral mucosa
that pathophysiology is complex and multifactorial [8].
Histopathological evaluation of mucositis lesions shows
mucosal thinning, caused by apoptosis and depletion of
the epithelial basal layer, with subsequent denudation and
secondary bacterial infection [9]. Development of oral
mucositis involves oxidative stress and the accumulation
of reactive oxygen species (ROS) [10]. This oxidative
stress can produce lipid peroxidation and inflammation
[11]. The inflammatory response in oral mucositis in-

O. M. de S. Sá et al. / Natural Science 5 (2013) 972-978 973
volves the activation of NF-κB and the upregulation of
inflammatory cytokines, including TNF-α [12] Glycine,
a simple amino acid, has been shown to have anti-in-
flammatory, immunomodulatory and cytoprotective ef-
fects [13]. Experimental studies have shown its protec-
tive effect on inflammatory lesions in various models.
For example, glycine is a strong inhibitor of resident liv-
er macrophages and acts via a glycine-gated chloride
channel, which subsequently inhibits Kupffer cell (KC)
activation by decreasing calcium inflow [14,15]. More-
over, glycine is an essential component of glutathione,
which is needed for detoxification processes. In addition,
glycine has indirect effects as a free radical scavenger
[16].
Glycine inhibits the production of inflammatory me-
diators, probably by decreasing the activation of NF-κB
and TNF-α, reducing the formation of additional free
radicals and other toxic mediators, and attenuating fur-
ther lipid peroxidation and glutathione depletion [17,18].
Recent research by Stoffels et al [19] has demonstrated
the ability of glycine to reduce chemotherapyassociated
injury. For example, in a clinically relevant in vivo model
of chemotherapyassociated liver injury, glycine decreased
liver damage (as measured by transaminases after chemo-
therapy), reduced microvesicular steatosis, and increased
hepatic microcirculation [19].
Further, in a rat model of ischemia-reperfusion injury,
glycine administration resulted in downregulation of
cell apoptosis and the expression of pro-apoptotic genes
[20]. Thus, glycine has been demonstrated to have po-
sitive effects on many of the pathways involved in the
pathogenesis of OM. Therefore, this study was designed
to assess the effects of glycine on chemotherapy-induc-
ed OM in a hamster model. We examined the effects of
systemic glycine supplementation on the clinical seve-
rity of OM, degree of inflammatory response (by assess-
ing neutronphil infiltrate histologically), and oxidative
stress (by measuring the final product of lipid peroxida-
tion).
2. MATERIALS AND METHODS
This study was approved by the Ethics Committee of
São Paulo Federal University (UNIFESP) (1916/08). Forty
female Golden Syrian hamsters (Mesocricetus auratus),
8 weeks old and weighing approximately 150 g each,
were used. The animals were kept in groups of six per
plastic container, with food and water available ad libi-
tum.
2.1. OM Induction Protocol
A well-accepted published protocol for chemotherapy-
induced oral mucositis in hamsters was used [21]. Briefly,
all the animals received 80 mg/kg intraperitoneally of the
chemotherapy drug 5-Fluorouracil (5-FU) on day 0, fol-
lowed by 40 mg/kg 5-FUadministered intraperitoneally
on day 2. The right cheek pouch of the animals was everted
and the mucosa was irritated by superficial scratching
with the tip of an 18-gauge needle by the same operator
on days 3 and 4.
2.2. Glycine Supplementation
The animals were randomly divided into two groups
of 20 animals each. Animals in Group 1 received a 2
mg/g of body weight intraperitoneal injection of Glycine
(Ajinomoto, Raleigh, NC), diluted in saline at a concen-
ration of 5%. Treatment with the Glycine, was initiated
on day 0, with application once per day (in the morning),
for seven days. Animals in Group 2 served as controls
and did not receive any glycine supplementation but were
treated identically in all other respects.
2.3. Clinical Evaluation of OM
Clinical evaluation of OM was performed by two blinded
evaluators. On day 3 and day 7, the right cheek pouch of
all animals was turned outward for the clinical evaluation
of the severity of the mucositis. Mucositis scores from 0
to 5 was assigned based on the method described by
Sonis et al with higher scores indicating greater severity
[22] (Table 1).
2.4. Histological Evaluation of OM
All animals were sacrificed on day 7 and the right
cheek pouch removed. The cheek pouch samples were
labeled, immediately cooled in isopentane for 10 s, and
then flash frozen in liquid nitrogen. The fragments were
positioned in such a way so as to provide cross-sectional
slices during microtomy. Serial slices (10 µm) were ob-
tained in a cryostat at a temperature of −20˚C, placed on
silanized glass slides, submerged in acetone, and dried at
room temperature for 10 min. The serial sections of each
sample were stained using hematoxylin-eosin staining
and examined under a light microscope by a blinded pa-
thologist. The absence or presence of microscopically
visible ulceration and severity of neutrophil infiltrate were
each separately scored, using the scale described by Lopes
et al. [23] (Table 1).
2.5. Measurement of Oxidative Stress:
Determination of Malondialdehyde
(MDA) Levels
MDA is a final product of lipid peroxidation and a
well-established measure of the level of free radicals in
intestinal tissue [20,24]. To determine MDA levels, the

O. M. de S. Sá et al. / Natural Science 5 (2013) 972-978
974
Table 1. Scales used for clinical and histological evaluation of oral mucositis.
GradeCriteria
0 Pouch completely healthy. No erosion or vasodilatation.
1 Erythema, but no evidence of mucosal erosion.
2 Severe erythema, vasodilation and superficial erosion
3 Formation of ulcers in one or more places, but not affecting more than 25% of the surface area of the pouch.
4 Severe erythema and vasodilation Cumulative ulcer formation about 50% of pouch surface area.
Clinical
Assessment
5 Virtually complete ulceration of the pouch mucosa. Loss of pliability.
0 Absent or rare neutrophil
Histological Assessment:
Neutrophil infiltrate 1 Moderate or severe neutrophil infiltrate
0 Ulceration absent
Histological Assessment:
Ulceration 1 Ulceration present
thiobarbituric acid (TBA) reaction proposed by Kohn
and Liversedge [25] was used. Tissue samples were de-
frosted, weighed, and a volume equivalent to five times
the weight of TRIS 0.01 M/pH 7.4 buffer solution was
then added. Tissue samples were homogenized in an ice
bath four times, for 30 seconds each, and subsequently
centrifuged for 5 minutes at 10,000 rpm, at 4˚C. The pro-
tein content of the homogenate was determined by the
coomassie brilliant blue (CBB) procedure, as described
by Kohn and Liversedge [25]. Briefly, the CBB reactant
interacts with protein, enabling its quantification by us-
ing a standard albumin curve with known concentrations.
Chi square test and the Fisher test. Quantitative variables
(MDA levels) were compared using the analysis of vari-
ance (ANOVA). All statistical analyses were performed
with a significance level of 5% (α = 0.05).
3. RESULTS
3.1. Clinical Evaluation of OM
There was excellent inter-examiner agreement on the
clinical assessment of OM (Kappa = 0.86 for Group 1
(glycine supplementation) and 0.94 for Group 2 (con-
trols). These data demonstrate that there was adequate
calibration for evaluation of the clinical characteristics of
OM. The mucositis induction protocol consistently caused
erythema, hemorrhage and ulceration in the right cheek
pouch of all animals. Thus, all animals in both groups
were scored as having Grade 3 mucositis on day 3 (Fig-
ure 1). However, by day 7, there was a marked reduction
in Clinical mucositis severity in most animals in the gly-
cine group, with the majority showing healing of ulcera-
tions. In comparison, the clinical mucositis severity in
control animals stayed the same or worsened (Ta b le 2 ).
This difference between groups was clinically and statis-
tically significant (p < 0.001).
For MDA measurement, 400 micro liters of the cen-
trifuged homogenate supernatant were collected and added
to 1 ml of 20% trichloroacetic acid and 400 ml of 1.6%
thiobarbituric acid.
The mixture was incubated for 30 minutes at 95˚C.
Lipids were extracted by adding n-butanol (1.6 ml) and
stirring vigorously. The sample was again centrifuged for
10 minutes at 3000 rpm.
Absorbance of the organic layer was determined through
reading at 510, 532, and 560 nm. The following equation,
proposed to minimize the interference of both heme
pigments and hemoglobin in the measurement of MDA
[20,24], was used:

MDA532
1.22A5320.56 A5100.44 A560 .  
 
3.2. Histological Evaluation of OM
Histopathological findings in control animals on day 7
were consistent with those previously described for this
animal model and mucositis induction protocol [22]. In
general, control animals demonstrated an intense cellular
infiltration with prevalence of neutrophils, hemorrhagic
areas, severe vascular hyperemia, edema, and ulceration.
Focal points of surface bacterial colonization and ab-
scesses were seen (Figure 2). In contrast, the glycine
group generally exhibited a less intensive histopathology-
cal reaction, with discreet vascular hyperemia and slight
inflammatory infiltration.
The calibration curve was drawn with 1, 3, 3 tetrameth-
oxypropane (also known as malondialdehyde bis. MDA
levels were calculated and expressed in nmol MDA/mg
of protein.
2.6. Statistical Analysis
The Kappa coefficient (k) was calculated to determine
inter-examiner agreement for clinical assessments of OM.
Qualitative variables (clinical and histological scoring of
mucositis severity) were compared using the Pearson’s On day 7, 100% of animals in the control group had a

O. M. de S. Sá et al. / Natural Science 5 (2013) 972-978 975
Figure 1. A representative Photographs of the cheek pouch of
hamsters at magnification ×400. Glycine group: (A) Day 3
(ulcer present) and (B) Day 7 (re-epithelization of the mucosa).
Control group representatives: (C) Day 3 (ulcer present) and (D)
Day 7 (persistent ulcer).
Table 2. Clinical evaluation of oral mucositis.
Day Group Mucositis grade
0 1 2 3 4 5
3 Glycine 20
Control 20
7 Glycine* 1 13 3 3
Control* 16 4
The table represents quantification of data represents number of animals
with each grade of oral mucositis at each time-point. *p < 0.001.
moderate-severe neutrophil infiltrate (grade 1), as com-
pared to only 25% of animals in the glycine group (Table
3). The remaining 75% of animals in the glycine group
demonstrated minimal neutrophil infiltrate (grade 0) (p <
0.001). On day 7, 100% of animals in the control group
demonstrated microscopic ulceration (grade 1), as com-
pared to only 35% of the animals in the glycine group
(Ta b l e 3). The remaining 65% of animals in the glycine
group demonstrated re-epithelization and healing, with
absence of microscopic ulceration (grade 0) (p < 0.001).
(a) (b)
(c)
Figure 2. A representative photomicrograph of hamster oral
mucosa on day 7 at magnification ×400, demonstrating ulce-
ration and inflammatory infiltration in epithelial cells. Glycine
group representative: (a) Absence of ulceration and inflamma-
tory infiltration. Control group representatives: (b) Moderate
inflammatory infiltration; (c) Intense inflammatory infiltration,
ulceration and bacterial colonization.
Table 3. Histological evaluation of oral mucositis (Day 7).
Neutrophil Infiltrate Ulceration
Group Grade 0*Grade 1* Grade 0# Grade 1#
Glycine 15 5 13 7
Control 0 20 0 20
The table represents quantification of data represents number of animals
with each grade of neutrophil infiltrate and ulceration, at day 7. *p < 0.001
#p < 0.001.
3.3. Measurement of Oxidative Stress:
Determination of Malondialdehyde
(MDA) Levels
At day 7, the mean MDA levels in the cheek pouch of
animals in the control group were more than 5-fold the
MDA levels in the glycine group (Table 4). Treatment
with glycine thus significantly reduced this marker of lipid
peroxidation and free radical production (p < 0.001).
4. DISCUSSION
OM is a complex process involving not only direct cell
injury caused by chemotherapy or radiation, but also a
complex cascade of biological events [10]. The process
begins with clonogenic cell death and the release of reac-
tive oxygen species (ROS), progressing through a series
of steps in which multiple biological pathways are acti-
vated and amplified, culminating in ulcer development,
and finally healing [4]. Investigations into the patho-

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976
Table 4. Measurement of oxidative stress: Determination of
malondialdehyde (MDA) levels (Day 7).
Nmol MDA/mg protein
Group Mean Standard DeviationN
Glycine 0.185* 0.118 20
Control 1.085* 0.225 20
*p < 0.001.
genesis of OM show the importance of the inflammatory
response, which includes the involvement of many dif-
ferent inflammatory mediators including nuclear factor
kappa B (NF-κB) [12,26,27], cytokines such as tumor
necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β),
interleukin-6 (IL-6) and platelet activating factor (PAF)
[1,22,28,29], as well as the cyclooxygenase pathway [1,
23,26,30].
Therefore, there has been significant interest in the
evaluation of anti-inflammatory strategies for the ame-
lioration of oral mucositis [31]. Glycine is a simple non-
essential amino acid that acts as an inhibitory neuro-
transmitter in the central nervous system (CNS), via a
glycine-gated chloride channel (GlyR) [32]. Outside of
the CNS, glycine had been presumed to be independently
biologically neutral for a long time, functioning only as a
building block for proteins. More recently, however, evi-
dence has accumulated indicating that glycine possesses
anti-inflammatory properties [19]. Our current results
support this anti-inflammatory role for glycine. In a well-
accepted animal model of chemotherapy-induced OM,
we found that glycine supplementation significantly re-
duced the severity of clinical mucositis. The attenuated
clinical severity was accompanied by a marked reduction
in neutrophil infiltrate, which suggests that glycine sup-
pressed the inflammatory response associated with mu-
cositis. Furthermore, glycine also reduced the production
of damaging free radicals, as measured by MDA levels.
MDA is a final product of lipid peroxidation and a well-
established measure of the level of free radicals in intes-
tinal tissue [20,24].
The anti-inflammatory effects of glycine are believed
to be mediated, at least in part, due to its mechanism of
action in the cell membrane where it activates the chlo-
ride channel that stabilizes or hyperpolarizes the mem-
brane potential [33]. Glycine blocks the increase of in-
tracellular calcium which stimulates the formation of the
cytokine cascade, inhibiting cells that activate the inflame-
matory process, probably by blocking activation of NF-
κB and TNF-α [34], decreasing the formation of free
radicals and other toxic mediators [35]. The free radical
nitric oxide and/or its derivatives can cause lipid peroxi-
dation [36], oxidation of protein sulfhydryls [37] and ni-
tration of tyrosine residues on a variety of proteins, in-
cluding inactivation of enzymes and/or receptors [38-40].
These effects result in tissue injury, which can lead to an
excessive local amplification of the inflammatory re-
sponse.
Mikalauskas et al. demonstrated that glycine decreas-
ed chemotherapy-induced liver injury, accompanied by a
significant reduction in inducible nitric oxide synthase
[19]. Our finding of reduced MDA levels and neutrophil
infiltrates in glycine-treated animals is consistent with a
role for glycine in reducing lipid peroxidation, free radi-
cal formation, and the subsequent tissue injuryand in-
flammatory response. In a rat model of rheumatoid ar-
thritis, glycine supplementation reduced joint swelling,
accompanied by a reduction in TNF-α, inflammatory cell
infiltrate and edema [41]. Furthermore, in a rat model of
ischemia-reperfusion injury, glycine administration re-
sulted in increased mucosal viability and thickness, likely
mediated via a down-regulation of cellular apoptosis [20,
42]. Another possible mechanism is derived from the fact
that glycine participates in the formation of a third of the
structure of collagen. Thus, glycine supplementation may
result in increased basement membrane stability [43].
Collectively, these studies point to several mechanisms
whereby glycine may be beneficial in ameliorating che-
motherapy-induced mucosal injury.
5. CONCLUSION
In conclusion, glycine supplementation significantly
reduced chemotherapy-induced oral, mucosal injury, neu-
trophil infiltrate and free radical production in an animal
model.
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