Mutagenesis, 2020, 35, 291–297

doi:10.1093/mutage/geaa012
Regular Article
Advance Access publication 22 April 2020

Regular Article

Advanced glycation end-products accelerate

telomere attrition and increase pro-inflammatory
mediators in human WIL2-NS cells

Downloaded from https://academic.oup.com/mutage/article/35/3/291/5823765 by guest on 31 October 2023

Permal Deo1,2,*, , Varinderpal S. Dhillon1,2,*, Wai Mun Lim1,
Emma L. Jaunay1,2, , Leigh Donnellan1,2, Brock Peake1,2,
Caitlin McCullough1 and Michael Fenech1,2,3
1
School of Pharmacy and Medical Sciences, University of South Australia, 108 North Terrace, Adelaide 5001, Australia,
2
Health and Biomedical Innovation, School of Pharmacy and Medical Sciences, University of South Australia, 61–68
North Terrace, Adelaide 5001, Australia and 3Genome Health Foundation, 25 King George Avenue, North Brighton
5048, Australia

*To whom correspondence should be addressed. School of Pharmacy and Medical Sciences, University of South Australia,
108 North Terrace, Adelaide 5001, Australia. Tel: +61 8 83021189; Fax: +61 8 83022389; Email: Permal.Deo@unisa.edu.au
Received 27 February 2020; Editorial decision 27 March 2020; Accepted 7 April 2020.

Abstract
This study investigated the effect of dietary sugars and advanced glycation end-products (AGE)
on telomere dynamics in WIL2-NS cells. Dietary sugars [glucose (Glu) and fructose (Fru); 0.1 M
each] were incubated with bovine serum albumin (BSA) (10 mg/ml) at 60 ± 1°C for 6 weeks to
generate AGE-BSA. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed total
AGE levels as 87.74 ± 4.46 nmol/mg and 84.94 ± 4.28 nmol/mg respectively in Glu-BSA and Fru-
BSA model. Cell treatment studies using WIL2-NS cells were based on either glucose, fructose
(each 2.5–40 mM) or AGE-BSA (200–600 µg/ml) in a dose-dependent manner for 9 days. Telomere
length (TL) was measured using qPCR. Nitric oxide (NO) production and tumour necrosis factor-α
(TNF-α) levels were measured in WIL2-NS culture medium. An increasing trend for TNF-α and NO
production was observed with higher concentration of glucose (R2 = 0.358; P = 0.019; R2 = 0.307;
P = 0.027) and fructose (R2 = 0.669; P = 0.001; R2 = 0.339; P = 0.006). A decreasing trend for TL
(R2 = 0.828; P = 0.000), and an increasing trend for NO production (R2 = 0.352; P = 0.031) were
observed with increasing Glu-BSA concentrations. Fru-BSA treatment did not show significant
trend on TL (R2 = 0.135; P = 0.352) with increasing concentration, however, a significant reduction
was observed at 600 µg/ml (P < 0.01) when compared to BSA treatment. No trends for TNF-α levels
and a decreasing trend on NO production (R2 = 0.5201; P = 0.019) was observed with increasing
Fru-BSA treatment. In conclusion, this study demonstrates a potential relationship between
dietary sugars, AGEs and telomere attrition. AGEs may also exert telomere shortening through
the production of pro-inflammatory metabolites, which ultimately increase the risk of diabetes
complications and age-related disease throughout lifespan.

Introduction in body fluids resulting in hyperglycaemia (1,2). If left untreated,

hyperglycemia among various pathways could lead to the formation
Diabetes mellitus (DM), a metabolic disorder, occurs when the pan-
and accumulation of advanced glycation end-products (AGEs) which
creas is unable to produce or secrete an effective amount of insulin,
includes Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine
or cells become insensitive to insulin. Therefore, glucose accumulates

© The Author(s) 2020. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
291
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292
(CEL), methylglyoxal derived hydroimidazolones and argpyrimidine
adducts (3–6). There has been a proposed link between accumu-
lation of AGEs in the body, especially in individuals with type 2
DM, and an increase in the level of oxidative stress (OS), inflam-
mation and genome damage. AGEs stimulate OS in different cells
by binding to receptor of AGEs (RAGEs), resulting in the activation
of NADPH oxidase, that leads to the production of reactive oxygen
species (ROS) (7). Furthermore, ROS could also activate transcrip-
tion factor, NF-κB which triggers a cascade signalling pathway and
increases expression of pro-inflammatory cytokines such as tumour
necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6 and inducible
nitric oxide (NO) synthase (8,9). Increased ROS levels have been re-
ported to activate numerous cell-signalling pathways related to cell
damage and apoptosis (10).
Telomere length (TL) is used as an indicator biomarker of
genome stability and disease risk (11,12). It has been reported that
elevated inflammatory activity could accelerate telomere attrition by
promoting cell turnover and replicative senescence (13). In vitro re-
search indicates that an accumulation of senescent cells with critic-
ally short telomeres may produce pro-inflammatory factors which in
turn increase inflammation load (14). Emerging evidence has shown
a direct relationship between increased OS and telomere dysfunction
(15). TNF-α and NO are often used as relevant biomolecules for
their role in inflammation and/or cell death induction (16). TNF-α
is involved in systemic inflammation, stimulation of acute phase re-
actions and induction of apoptotic cell death (16,17). NO has been
implicated in cell injury caused by activated microglia and induction
of DNA damage in pancreatic β cells which may result in reduced
insulin secretion (18). Current literature suggests that diabetes is one
of the risk factors that leads to telomere shortening, evident in mono-
cytes and leukocytes (11,19). Hyperglycaemia creates a high OS con-
dition due to ROS overproduction, which in turn leads to oxidative
DNA damage and results in telomere shortening and/or DNA breaks
leading to telomere loss (20). Eventually, these conditions could lead
to cell senescence and increases in cell turnover rate (11).
Several factors may cause genome damage and cell apoptosis,
including dietary sugars, endogenous AGEs and OS (21–23). OS has
been proposed as the main factor causing telomere shortening and
can trigger senescence (24). We have recently reported the role of
reducing sugar and related AGEs on increased chromosomal DNA
damage (25). The present study is aimed at testing the hypotheses
that (i) dietary sugars and AGEs accelerate telomere attrition and
(ii) the production of inflammatory cytokines may be one of the
mechanisms contributing to telomere shortening in WIL2-NS cells
induced by sugars and AGEs.
Materials and Methods
Preparation of endogenous AGE models and
quantification
A reducing sugar-induced protein glycation model was used to pre-
pare AGEs, as previously described (25). Briefly, bovine serum al-
bumin (BSA, 10 mg/ml, final concentration, Sigma-Aldrich, Sydney,
Australia) was mixed with either 0.1 M glucose (Glu-BSA, Sigma-
Aldrich) or 0.1 M fructose (Fru-BSA, Sigma-Aldrich) in 0.2 M
phosphate buffer (pH 7.4) and incubated at 60°C for 6 weeks. BSA
(10 mg/ml) only was used as a control. After incubation unreacted
sugars and endotoxins were removed from the incubated samples as
previously described (4).
For protein-bound CML and CEL levels, glycated samples were
prepared using acid hydrolysis technique previously described
P. Deo et al., 2020, Vol. 35, No. 3
(4,26). For protein-bound methylglyoxal derived, hydroimidazolone
1 (MG-H1) samples were prepared by enzymatic hydrolysis as pre-
viously described (27). Analysis of CML, CEL and MG-H1 were
performed using a Sciex QTRAP 6500+ liquid chromatography-mass
spectrometer (Sciex, Framingham, MA, USA) and detected in ESI
positive multiple reaction monitoring (MRM) mode as described
previously (25).
WIL2-NS cells and cell culture
The WIL2-NS cell line was selected as it is commonly used in studies
of DNA damage and telomere defects induced by OS and nutritional
factors (28–31). WIL2-NS cells are human spleen B lymphoblastoid
cells that have suppressed apoptotic activity and high nuclear div-
ision index (NDI), as a result of a G-A transition mutation in codon
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237 of the p53 gene allowing survival of cells with genome damage
by suppressing apoptosis (32,33).
WIL2-NS cells were cultured in RPMI 1640 complete medium
(Sigma, Sydney, Australia), supplemented with 5% (v/v) sterile
foetal bovine serum (FBS) (Thermo Trace, Melbourne, Australia),
1% (v/v) penicillin [5000 IU/ml]/streptomycin [5000 µg/ml] (Sigma,
Australia) and 1% (v/) 200 mmol/l L-glutamine (Sigma, Australia)
at 37°C in a 5% CO2 humidified incubator (Quantum Scientific,
Brisbane, Australia). Cells were cultured in 24-well plates (Themo
Fisher Scientific, NY, USA) at an initial density of 2 × 103 cells/ml and
the medium replaced every 3 days. Cell count was performed using
a Coulter Counter (Beckman Coulter, Z1 Coulter Particle Counter,
USA) and viability test was carried out using 0.4% Trypan Blue solu-
tion assay (Sigma, Australia).
Study design and treatment
WIL2-NS cells (2 × 106 cells/ml) were treated with either the redu-
cing sugar or AGEs, as recently reported (25). For reducing sugar
study, cells were treated with either glucose (2.5–40 mmol/l), or
fructose (2.5–40 mmol/l) in glucose-depleted RPMI 1640 medium
supplemented with 5% (v/v) FBS, 1% (v/v) penicillin [5000 IU/ml]/
streptomycin [5000 µg/ml], 1% (v/) 200 mmol/l L-glutamine and
2 g/l sterile sodium bicarbonate (Sigma, Australia). For AGEs study,
cells were treated either with BSA (200–600 µg/ml), Glu-BSA (200–
600 µg/ml) or Fru-BSA (200–600 µg/ml) in RPMI 1640 complete
media (containing approximately 10 mM glucose). All cells were
cultured at 37°C in a 5% CO2 humidified incubator for 9 days with
medium change every 3 days. After treatment, cells (cell count: 2.2 ×
106 − 4 × 106) were harvested and stored at −80°C until use.
DNA isolation and Real-Time qPCR assay for TL
measurement
Genomic DNA was extracted from isolated lymphocytes using the
QIAamp DNA blood mini kit (Qiagen, Australia). Purified DNA
samples were quantified using NanoDrop 1000 spectrophotometer
(Thermo Fisher Scientific, Australia) and diluted as per experimental
requirements (5 ng/µl). TL was measured using quantitative real-
time PCR, as described previously (34). The ratio of the telomere
(T) repeat copy number to the single-copy gene (S) was determined
for each sample using ABI 7300 Real-Time PCR Detection System
(Life Technologies, USA). The final concentrations of the PCR re-
agents were 1 × SYBR Green Mix (Life Technologies, USA), 20 ng
DNA, 0.2 µmol of telomere specific primers (F: 5′-GGTTTTTGAG
GGTGAGGGTGAGGGTGAGGGTGAGGGT-3′; R: 5′-TCCCGAC
TATCCCTATCCCTATCCCTATCCCTATCCCTA-3′) and 0.3 μmol
of 36B4 primers (F: 5′-CAGCAAGTGGGAAGGTGTAATCC-3′; R:
AGEs accelerate telomere attrition, 2020, Vol. 35, No. 3
5′-CCCATTCTATCATCAACGGGTACAA-3′). The reactions were
performed using telomere and 36B4 specific primers in a 96-well
plate, and each plate included a reference DNA sample. A five-point
serial dilution standard curve of DNA concentration versus T/S ratio
using DNA isolated from the 1301 cell line (which has a mean TL of
23 000 base pairs) was established in each plate. The standard curve
was then used to convert the T/S ratio into TL in base pairs (bp)
using following equation: Absolute TL (bp) = 2433.23X + 3109.51
where X = T/S ratio, 2433.23 is the slope and 3109.51 is the inter-
cept of the standard curve. A standard curve with a high correlation
factor (R2 > 0.97) was required to accept the results from the plate.
We calculated intra-assay and inter-assay CVs to assess the variation
of results within the data set and to ensure plate-to-plate consist-
ency, respectively. The intra-assay coefficient between duplicates was
2.3% for telomeres and 2.1% for the single-copy gene, whereas the
inter-assay CV between plates was 0.5% for telomeres and 0.88%
for the single-copy gene.
Determination of and TNF-α and NO levels
The TNF-α levels in the spent media, released by the WIL2-NS cells
during treatments was measured using ELISA (Product: EK-0001;
Elisakit, Melbourne, Australia) using the manufacturer’s instruc-
tions. The captured antibody in the wells were activated by adding
assay diluent 1B into each well, followed by adding zero samples,
standards and samples into the allocated wells. A standard curve
ranging from 31.25 to 200 pg/ml was generated. After washing, the
detection of TNF-α was done by adding biotin labelled detection
antibody, the conjugation of streptavidin-HRP conjugate and sub-
strate as the detection agent. The plate was incubated for 15 min in
dark to develop the blue colour. The reaction was stopped by add-
ition of stop solution. Absorbance was measured at 450 nm using a
multi-scan agent reader (Thermo Fisher, Australia).
NO levels in the spent media were determined by Griess reagent.
Spent media (70 µl) and Griess reagent (70 µl, Sigma, Australia) were
aliquoted in triplicate into a 96-well plate and the mixture was in-
cubated at 25 ± 1°C with shaking for 15 min. The absorbance of
samples was measured at 540 nm using a multi-scan agent reader
(Thermo Fisher, Australia). The level of NO was calculated by com-
paring to a sodium nitrite standard curve prepared in PBS (pH 7.4)
ranging from 0 to 100 µM.
Statistical analysis
For TL, analysis was carried in duplicate from three independent
experiments within each of the treatment (n = 3). For TNF-α and
NO, analysis was carried out in triplicates from three independent
experiments within each treatment (n = 3). The results are ex-
pressed as mean ± standard error of the mean (SEM). Parametric
or nonparametric tests were used depending on whether the results
had a normal Gaussian distribution or a non-Gaussian distribution,
respectively. One-way analysis of variance (ANOVA) followed by
multiple comparison tests was used to determined using GraphPad
Prism 8 (San Diego, CA, USA). Trend tests for dose responses were
also performed. Correlation analysis was performed by Pearson’s (rp)
test. Statistical significance was accepted for values P < 0.05.
Results
AGE levels in reducing sugar models
Total AGE levels in Glu-BSA model was 87.74 ± 4.46 nmol/mg BSA
(containing 26.76 ± 1.09 nmol CML/mg BSA; 5.41 ± 0.05 nmol
293
CEL/mg BSA; 55.57 ± 3.32 nmol MG-H1/mg BSA), whereas Fru-
BSA model showed 84.94 ± 4.28 nmol/mg BSA (containing 7.30 ±
0.35 nmol CML/mg BSA; 7.87 ± 0.19 nmol CEL/mg BSA; 69.77 ±
3.74 nmol MG-H1/mg BSA). Total AGE concentration in con-
trol BSA was only 1.35 ± 0.07 nmol/mg BSA (containing 0.56 ±
0.02 nmol CML/mg BSA; 0.22 ± 0.00 nmol CEL/mg BSA; 0.05 ±
0.05 nmol MG-H1/mg BSA).
Effect of dietary sugars on TL, TNF-α levels and NO
production
A 9-day treated WIL2-NS cells did not show any trend in TL either in
the glucose (R2 = 0.037; P = 0.175, Figure 1A) or fructose treatment
(R2 = 0.013; P = 0.644, Figure 1A). However, an increasing trend
for TNF-α level was observed with increased glucose (R2 = 0.358;
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P = 0.019, Figure 1B) and fructose (R2 = 0.669; P = 0.001, Figure 1B)
concentrations. Similarly, an increasing trend for NO produc-
tion was observed with increased glucose (R2 = 0.307; P = 0.027,
Figure 1C) and fructose (R2 = 0.339; P = 0.006, Figure 1C) con-
centrations. In glucose treatment, a 1-way ANOVA test showed sig-
nificant increase in TNF-α levels (R2 = 0.538; P = 0.048) and NO
production (R2 = 0.546; P = 0.041), respectively. In fructose treat-
ment, a 1-way ANOVA test showed significant increase in TNF-α
levels (R2 = 0.683; P = 0.014) and NO production (R2 = 0.721;
P = 0.008), respectively.
Effect of AGE-BSA on TL, TNF-α levels and NO
production
A decreasing trend for TL (R2 = 0.828; P = 0.001, Figure 2A), and
an increasing trend for NO production (R2 = 0.352; P = 0.031,
Figure 2C) were observed with increasing Glu-BSA concentrations.
No trend for TNF-α levels (R2 = 0.219; P = 0.242, Figure 2B) with
increased Glu-BSA concentration was observed. Glu-BSA treatment
showed a significant reduction in TL at 400 µg/ml (P < 0.05) and
further reduced when the concentration increased to 600 µg/ml
(P < 0.001) compared to BSA only treatment. Furthermore, Glu-BSA
treatment showed significant increase in TNF-α levels at 400 µg/ml
(P < 0.01) and 600 µg/ml (P < 0.01), respectively. NO production
only showed a significant increase at 600 µg/ml (P < 0.01) when
compared to BSA only treatment. In the Glu-BSA treatments, a sig-
nificant negative correlation was observed between TL versus TNF-α
(rp = −0.622, P < 0.05) and NO (rp = −0.700, P < 0.05), respectively
(Table 1).
Fru-BSA treatment did not show a significant effect trend on
TL (R2 = 0.135; P = 0.352; Figure 2A) with increasing concentra-
tion however, a significant reduction was observed at 600 µg/ml
(P < 0.01) when compared to BSA only treatment. In addition, no
trends for TNF-α levels (R2 = 0.015; P = 0.773; Figure 2B) was ob-
served with increasing Fru-BSA treatment. Interestingly, a decreasing
trend on NO production (R2 = 0.521; P = 0.019; Figure 2C) with
increasing Fru-BSA concentrations was evident. BSA only treat-
ment did not show any significant effect trends on TL (R2 = 0.019;
P = 0.747), TNF-α levels (R2 = 0.091; P = 0.408) and NO pro-
duction (R2 = 0.316; P = 0.118) with increasing concentrations
(Figure 2A–C).
Discussion
Previous studies have shown that reducing sugars, including glu-
cose and fructose, contribute to the formation of AGEs and
create excessive OS in the body (9,21,35). Telomere defects are
294
Fig. 1. Effect of dietary sugar on telomere length (A), tumour necrosis factor-
α (B) and nitric oxide production (C) in WIL2-NS cells. Results are expressed
as mean ± SEM, n = 3 and values with different superscript letters are
significantly different from each other (P < 0.05).
measured as telomere shortening, telomere loss, anaphase bridges
or nucleoplasmic bridges (NPB) originating from telomere end-to-
end chromosome fusions all of which have been associated with in-
creased risk of age-related diseases (33,36–38). Evidence regarding
the relationship among hyperglycaemia, Hb1Ac on TL have been
reported (1,39–41) and a meta-analysis of human studies reported a
significant TL shortening in subjects with diabetes related to healthy
controls (42). However, the effect of sugars, particularly fructose on
P. Deo et al., 2020, Vol. 35, No. 3
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Fig. 2. Effect of AGEs on telomere length (A), tumour necrosis factor-α (B)
and nitric oxide production (C) in WIL2-NS cells. Results are expressed as
mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 when compared with
the respective control.
TL, and the relationship between AGEs and genomic damage is still
lacking. Thus, this study investigated the effect of sugars and related
AGEs on TL in a dose-dependent response.
We chose a leukocyte cell line because several studies show a con-
sistent correlation between a higher risk of diabetes and telomere
shortening in leukocytes (43). Furthermore, two prospective studies
were reported recently showing that telomere shortening in leukocytes
predicted a higher risk of diabetic kidney disease progression and is-
chaemic heart disease in patients with type 2 diabetes (44,45). Glucose
and fructose were chosen as the reducing sugars for testing due to their
increasing abundance in Western diets and their high level of reactivity
in AGE formation. Dietary sugar concentration (2.5–40 mmol/l) was
chosen to represent levels found in subjects who are non-diabetic
AGEs accelerate telomere attrition, 2020, Vol. 35, No. 3
Table 1. Pearson’s correlation among Telomere length, tumour
necrosis factor-α and nitric oxide production in sugars and re-
lated AGEs
Pearson correlation (rp) Telomere length
Glucose Fructose BSA Glu-BSA Fru-BSA
TNF-α 0.155 −0.042 −0.375 −0.622* −0.149
NO −0.214 −0.372 0.219 −0.700* 0.232
*P < 0.05.
(low/normal; 2.5–10 mmol/l), diabetic (high; >10 mmol/l) and chronic
hyperglycemia (40 mmol/l) (46,47). Glucose levels ranging from 33.3
to 45 mmol/l as chronic treatment have been previously reported in
cell studies showing cellular dysfunctions and apoptosis related to type
2 diabetes (47,48). A dose-dependent concentration (200–600 µg/ml)
was explored for AGEs Glu-BSA and Fru-BSA to cover a low-high
range representing normal to diabetic conditions. A previous study
used Glu-BSA ranging from 10 to 500 µg/ml in cell treatment for
28 days to represent diabetic models (49). We chose 9 days because in
our previous study with the WIL2-NS model we showed that reducing
sugars and/or their AGEs induce micronuclei, nucleoplasmic bridges
and nuclear buds that are biomarkers of telomere loss which could
be due to terminal chromosome fragment loss, telomere dysfunction
observed as dicentric chromosomes anaphase bridges due to telomere
end-fusions, and extrusion of amplified gene sequences generated by
repeated cycles of breakage and fusion of the dicentric chromosomes,
respectively (25,37).
In this study, neither glucose nor fructose showed any signifi-
cant effect on TL. However, in the AGE treatments, Glu-BSA treat-
ment showed a significant decreasing trend in TL, whereas Fru-BSA
showed a non-significant trend for TL with AGE dose. However, at
600 µg/ml Fru-BSA, a 1-way ANOVA showed a significant difference
in TL when compared to controls. We recently reported that fruc-
tose treatment exerted a significant genotoxic dose-response effect
in WIL2-NS cells measured using the cytokinesis block micronucleus
cytome assay (CBMNcytome assay (25)), whereas, DNA damaging
effects of glucose were less evident. In the same study, high concen-
trations of AGEs (400–600 µg/ml) induced DNA damage; however,
cytotoxicity (necrosis and apoptosis) was not affected (25). The bio-
markers of DNA damage in the CBMNcytome assay indicate the
possibility of chromosome breakage and/or whole chromosome
loss (micronuclei) and DNA misrepair and/or telomere end-fusions
(nucleoplasmic bridges) and gene amplification (nuclear buds) which
all are key aspects of chromosomal instability that are mechanistic-
ally associated with telomere shortening and/or dysfunction (28,37).
The telomere shortening induced by Glu-BSA is consistent with the
dose-related induction of this AGE class with micronuclei (MNi),
nucleoplsamic bridges (NPB) and nuclear buds (Nbuds) in our
earlier study using the same model (25). Despite the lack of research
on dietary sugars and/or related AGEs, and TL, several human
studies have published an association between insulin resistance,
HbA1c and telomere shortening (50–53). A study showed significant
difference in the mean leukocyte TL logarithm results by comparing
to the control group (n = 424) who had neither first-degree relatives
with diabetes nor glucose intolerance and diabetic group (n = 432)
(P = 0.003) (54). In addition, a human study has reported an as-
sociation between leukocyte TL and diabetic complication by com-
paring the control subjects (n = 400) and diabetes patients (n = 501)
(P < 0.001) (51). Recently, Grunnet and colleagues (53) showed a
significant negative association between TL and fasting glucose
295
(P = 0.003) and HbA1c (P = 0.0008), respectively. Sampson and col-
leagues reported that telomere lengthening is negatively correlated
with levels of 8-hydroxy deoxyguanosine, a biomarker of oxidative
DNA damage, in patients with DM (19). Three recent meta-analysis
have also demonstrated a significant association between a short-
ened TL and risk of DM (42,50,52).
In the present study, we also included TNF- α and NO as meas-
ures of inflammatory activity. These markers were significantly posi-
tively correlated with each other in the glucose (rp = 0.685, P < 0.01),
fructose (rp = 0.510, P = 0.05) and Glu-BSA (rp = 0.469, P = 0.05) but
not in the Flu-BSA (rp = −0.126, P = 0.746) treatment. The increased
levels of these markers in the medium may reflect different aspects
of the inflammatory response. Therefore, it is possible that TNF- α
and NO may contribute to telomere shortening by promoting cell
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turnover and replicative senescence, including OS and regulating tel-
omerase activity (14,55,56). Furthermore, chronic exposure to the
cumulative load of both high TNF-α and NO may have greater im-
pact on telomere attrition. Both TNF-α and NO production showed
an increasing trend when WIL2-NS cells were exposed to either glu-
cose or fructose in our study. In addition, a significant increase in the
levels of TNF-α and NO were observed at the higher concentration
of sugar treatment (40 mM), indicating the adverse metabolic impact
caused by high concentrations of dietary sugars. A previous study
demonstrated that high glucose condition (50 mM) can significantly
induce the production of TNF-α (P < 0.05) (57). In the present study,
TNF-α levels were significantly higher in the Glu-BSA treatment but
not in the Fru-BSA treatment. Similarly, NO levels produced in the
Glu-BSA treatment were significantly higher (P < 0.01) when com-
pared with the control. TL was negatively correlated with TNF-α
and NO in the Glu-BSA treatment, but this was not observed in any
other treatments in this study. Previous investigations have shown a
dose-dependent increase in NO, TNF-α levels and intracellular ROS
formation when cells were exposed to AGE-BSA (58,59). Another
study showed that cells exposed to AGE-modified-compounds sig-
nificantly increased TNF-α levels (P < 0.05) when compared to the
control (4). These results suggest an association between AGE-BSA
exposure and the production of ROS, possibly in a dose-dependent
manner. A study found a negative correlation between the production
of ROS and TL, which could lead to the development of senescence
(60). Furthermore, it is known that TL is maintained by telomerase
and regulated by pro-inflammatory cytokines and OS (61). Taken
together, our results support the hypothesis that an increase in AGEs
increases the production of TNF-α levels and NO production and
cause an increased OS may accelerate telomere shortening.
In conclusion, our results showed that dietary sugars (glucose
and fructose) have less impact on telomere shortening compared
to their respective AGEs, where, Glu-BSA and higher concentra-
tion of Fru-BSA caused a significant telomere shortening. These
results suggest that AGEs play an important role in chromosomal
instability by damaging telomere maintenance. The production of
pro-inflammatory biomarkers (TNF-α, NO) was consistent with
the results of chromosomal DNA damage and telomere shortening
suggesting a plausible mechanistic association. These findings pro-
vide some new information on how production of AGEs may be a
genotoxic risk factor for the acceleration of degenerative disease in
humans.
Funding
The research was supported by the University of South Australia Teaching and
Research grant. E.L.J. and L.D. holds an Australian Government Research
Training Program Scholarship.
296
Acknowledgments
The authors acknowledge Maulik Ghetia for their technical assistance on
LC-MS/MS.
Conflict of interest statement: None declared.
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