Effect of Lutein (Lute-gen®) on proliferation rate and telomere length in vitro and possible mechanism of action

Abstract

Background: Many studies have reported that lutein could exert its biological activities, including anti-inflammation, anti-oxidative and anti-apoptosis, through its effect on reactive oxygen species (ROS). Thus, lutein may prevent the damaging effect of ROS in cells. Telomere length is one of the most important biomarkers of aging. It is known that oxidative stress can accelerate telomere shortening, whereas antioxidants like lutein can delay their attrition through their antioxidant activity. Aim of the study: This study was conducted to assess the effect of Lute-gen® (Lutein) on telomere length and cellular proliferation rate in cultures of human adult primary fibroblast cells grow under standard or oxidative stress conditions. Method: The current study investigated the effect of Lute-gen® on telomere length. For this purpose, a primary cell line was treated with different concentrations of Lute-gen® (10; 5; 1 μg/ml) in the presence or absence of the pro-oxidant hydrogen peroxide (H 2 O 2). Different concentrations of lutein were included along with positive (H 2 O 2) and negative (no treatment) controls. Determination of the telomere shortening rate along with the evaluation of the median telomere length, 20 th percentile length and the percentage of telomeres below 3 kilo base pairs (Kbp) was performed using an optimized, analytically validated HT-Q-FISH methodology. Results: Lute-gen was observed to exert a significant protective effect on telomere length erosion in vitro under oxidative stress conditions after eight passages. Conclusions: Lute-gen's effect on telomere attrition indicates that the antioxidant activity of lutein is beneficial and counteracts the effects of oxidation on telomeres in human primary cells. The results grant further investigation in vivo, on a controlled human clinical trial context. The use of in vitro modelling is beneficial to investigators developing natural products with anti-oxidant claims such as lutein and their effect on ageing. Introduction In human populations, telomere length is a biomarker of aging for a whole organism and a biomarker of aging in specific tissues [1]. The contribution to oxidation and/or inflammation to telomere shortening and hence aging requires longitudinal studies in which individuals, monitored over decades have to be followed. This approach requires time course determination of telomere length, in well-characterized populations that measure social, behavioural, medical, and biological factors. As cells age, they lose a certain number of base pairs, it is estimated that roughly 50 base pairs are lost during cell division because of the end-replication problem [15]. When telomeres have decreased to a critical length, cell division ceases, although cell senescence may continue for a time. This finite ability to replicate is known as the "Hay flick limit," and has been seen in normal cultured mammalian cells. Summarily, each time a cell divides, telomeres get shorter. When they get too short, the cells become "senescent" and eventually dies. Cell senescence is associated with aging and this could be due to ROS or oxidative stress [2, 5] .

Full text

International Journal of Biology Research
84
International Journal of Biology Research
ISSN: 2455-6548; Impact Factor: RJIF 5.22
Received: 19-06-2019; Accepted: 22-07-2019
www.biologyjournal.in
Volume 4; Issue 4; September 2019; Page No. 84-91
Effect of Lutein (Lute-gen®) on proliferation rate and telomere length in vitro and possible
mechanism of action
S Mehkri1, Diego Perez2, Pilar Najarro3, Menelaos Tsapekos4, KN Bopanna5*
1 Bio-gen Extracts Pvt. Ltd., 57, Sompura Industrial Area, Dobaspet, Bangalore, Karnataka, India
2, 3, 4 Life Length, C/Miguel Ángel, 11, Madrid, Spain
5 Consultant Pharmacologist, 9b Sobha Emerald Jakkur, Bangalore, Karnataka, India
Abstract
Background: Many studies have reported that lutein could exert its biological activities, including anti-inflammation,
anti-oxidative and anti-apoptosis, through its effect on reactive oxygen species (ROS). Thus, lutein may prevent the damaging
effect of ROS in cells.
Telomere length is one of the most important biomarkers of aging. It is known that oxidative stress can accelerate telomere
shortening, whereas antioxidants like lutein can delay their attrition through their antioxidant activity.
Aim of the study: This study was conducted to assess the effect of Lute-gen® (Lutein) on telomere length and cellular
proliferation rate in cultures of human adult primary fibroblast cells grow under standard or oxidative stress conditions.
Method: The current study investigated the effect of Lute-gen® on telomere length. For this purpose, a primary cell line was
treated with different concentrations of Lute-gen® (10; 5; 1 μg/ml) in the presence or absence of the pro-oxidant hydrogen
peroxide (H2O2). Different concentrations of lutein were included along with positive (H2O2) and negative (no treatment)
controls.
Determination of the telomere shortening rate along with the evaluation of the median telomere length, 20th percentile length
and the percentage of telomeres below 3 kilo base pairs (Kbp) was performed using an optimized, analytically validated HT-
Q-FISH methodology.
Results: Lute-gen was observed to exert a significant protective effect on telomere length erosion in vitro under oxidative
stress conditions after eight passages.
Conclusions: Lute-gen’s effect on telomere attrition indicates that the antioxidant activity of lutein is beneficial and
counteracts the effects of oxidation on telomeres in human primary cells. The results grant further investigation in vivo, on a
controlled human clinical trial context. The use of in vitro modelling is beneficial to investigators developing natural products
with anti-oxidant claims such as lutein and their effect on ageing.
Keywords: oxidant, counteracts, modelling, telomeres
Introduction
In human populations, telomere length is a biomarker of
aging for a whole organism and a biomarker of aging in
specific tissues [1].
The contribution to oxidation and/or inflammation to
telomere shortening and hence aging requires longitudinal
studies in which individuals, monitored over decades have
to be followed. This approach requires time course
determination of telomere length, in well-characterized
populations that measure social, behavioural, medical, and
biological factors.
As cells age, they lose a certain number of base pairs, it is
estimated that roughly 50 base pairs are lost during cell
division because of the end-replication problem [15]. When
telomeres have decreased to a critical length, cell division
ceases, although cell senescence may continue for a time.
This finite ability to replicate is known as the “Hay flick
limit,” and has been seen in normal cultured mammalian
cells. Summarily, each time a cell divides, telomeres get
shorter. When they get too short, the cells become
“senescent” and eventually dies. Cell senescence is
associated with aging and this could be due to ROS or
oxidative stress [2, 5].
In vitro studies conducted in primary cells to determine the
role of lutein (xanthophyll carotenoid) and its action on
telomere length are required to investigate its mechanism of
action. Here we focus on Lute-gen® and its effect on ROS.
In order to establish the effect of oxidative stress induced in
the presence of hydrogen peroxide was used as a pro-
oxidant. Cellular proliferation rate and telomere length were
measured in cultures of human adult primary fibroblast cells
treated with or without lutein under both standard and
oxidative stress conditions.
Materials and methods
Lute-gen® (Lutein) is manufactured from the Tagetes
erecta species of non-GMO marigold flowers using a
patent-pending manufacturing process. Lute-gen® is
standardized for Lutein content by HPLC and is
manufactured in a GMP, ISO and HACCP certified
manufacturing facility. The formulation of Lute-gen is done
with the use of natural ingredients only, including natural
vitamin E from sunflower that serves as an antioxidant. The
ingredient is Kosher and Halal certified, free of residual
solvents, soy-free and dairy-free, thereby making it suitable
for all population groups.

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Telomere analysis technology (TAT®)
All telomere length measurements were performed using
Life Length’s proprietary technology TAT. TAT is a robust
and reproducible high-throughput quantitative fluorescent in
situ hybridization (HT Q-FISH) technology that allows
measuring telomere length in individual chromosomes in
interphase cells. TAT has significant advantages over other
telomere testing methodologies. TAT is able to determine
telomere length in absolute units (base pairs – bp), report the
distribution and 20th percentile of telomere length and
inform of the % of short telomeres, allowing a more
comprehensive analysis of each sample. Each test sample is
plated in 5 replicates per plate to achieve statistical
significance. Fine tuning of the methodology has
accomplished high level of reproducibility; CV < 5% in
inter-plate repetitions.
Cell culture
Primary dermal fibroblasts; Normal, human, adult (HDFa)
(ATCC® PCS-201-012™) were used for this study. Cells
were seeded at 2x103 cells/cm2 in Gluta Max TM high
glucose Dulbecco's modified Eagle's medium (DMEM,
Gibco.) and supplemented with 10% fetal bovine serum
(FBS, HyClone, Thermo Scientific), penicillin (100 U/ml)
and streptomycin (1000 U/ml). Media was renewed every 2-
3 days and cells passaged at sub-confluence (70-80%) every
seven days. Cell growth was monitored for each condition
by counting cell numbers at each passage using a
Countess™ cell counter (Invitrogen). Population doubling
(PD) was calculated with the formula PD = (log
(Nn/Nn−1))/log 2 where n is the passage; and N the number
of cells. One PD is equivalent to one round of cell
replication. After passage four and eight, cells treated with
Lute-gen® ® and controls were frozen in liquid nitrogen
until their use.
Treatments
Human primary fibroblasts were treated with Lute-gen®
during eight weeks at three different concentrations (10, 5
and 1 μg/ml) under standard and oxidative (10 μM H2O2)
cell culture conditions. Fresh treatments were prepared and
added to the cells at every passage and also when media was
renewed. Stock solution of 10 mg/ml of the compound was
prepared in DMSO and the different treatments were
prepared from that stock. The final concentration of DMSO
was 0.5% at which no detrimental effect on cell growth or
toxicity was detected (data not included).
HT - Q Fish
Cell were thawed at 37o C and cell counts and cellular
viability were determined. Cells were seeded in clear bottom
black-walled 384-well plates at 12,000 cells per well in five
replicates for each sample and eight replicates for each
control cell line. Telomere length was measured by the High
Throughput Quantitative Fluorescence In Situ Hybridization
(HT-Q-FISH) technology (Canela et al., 2007) [6] on
interphase nuclei. The assay was conducted according to
quality standards of the Clinical Laboratory Improvement
Amendments (CLIA) and ISO15189. Each telomere is
hybridized with a fluorescent telomeric probe (PNA) that
recognizes a fixed number of telomeric sequences (bp). The
fluorescence intensity of the telomeric probes is directly
proportional to telomere length.
Image acquisition and processing
The OPERA High Content Bioimager (Perkin Elmer) was
used for automated image acquisition in combination with
the acapella software, version 1.8 (Perkin Elmer). Images
were captured, using a 40 x 0.95 NA water immersion
objective. UV and 488 nm excitation wavelengths were used
to detect the DAPI and A488 signals respectively. With
constant exposure settings, 15 independent images were
captured at different positions for each well. Cells were
stained with DAPI to facilitate autofocus of the microscope
and to define the region of interest for each cell, measuring
telomere fluorescence intensity of the A488 image in each
one.
Data analysis
An image algorithm was applied to allow cell nucleus
segmentation based on a local threshold. The intensity
results for each foci were exported to the Columbus 2.4
software (Perkin Elmer).
As mentioned above the fluorescence intensity from the
telomeric PNA probe that hybridize to a given telomere is
proportional to the length of that telomere. These intensities
were translated to base pairs through a standard regression
curve generated using control cell lines of known telomere
length.
Quality control analysis in each run includes an intra-plate
standard curve and 5 replicates per sample. The coefficient
of variation (CV) of the technique is < 5%.
Results
Proliferative Analysis
In order to determine the effect of Lute-gen on cellular
proliferation human primary fibroblasts were treated with
Lute-gen® at three different concentration (10, 5; and 1
µg/ml) during a period of eight weeks. Cumulative
population doubling (CPD) in standard conditions did not
present differences between the control group and groups
treated with Lute-gen®, except in the case of cells treated at
5 µg/ml, that showed a lower CPD compared to the rest of
the groups after six weeks (Fig 1).

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Fig 1: Growth curves of human adult primary fibroblasts untreated (ctrl-DMSO), treated with Lutein (10.0, 5.0 and 1.0 µg/m) under standard
and oxidative (10µM H2O2) condition. Each point on the population curve represents one passage.
Telomere Length measurements - Standard Conditions
A time course assay was conducted such that fibroblast cells
expanded under standard conditions as described in the
method section were detached from the cell culture flasks at
zero and then after four weeks and eight weeks. Samples
were frozen and analyzed at the end of the incubation
period. Data obtained following HT-Q-FISH analysis for
each of the variables determined are presented in the graphs
in Figure 2.
Compared to control attrition rates, after 4 weeks of
treatment with lutein at different concentrations we
observed a, statistically significant shortening in the median
telomere length variable for group treated with Lute-gen® at
1 µg/ml. Additionally, significant telomere shortening was
identified in the 20th percentile length variable, between the
control group and the group treated with Lute-gen® at 5
µg/ml.
Following 8 weeks of treatment with lutein at 10 and 1
µg/ml, statistically significant differences were detected in
the 20th percentile length as well as in the % of telomeres <
3 kbp variables when compared to the control group. The
statistical analysis and their p-values are summarized in
Tables 1-3.
Fig 2: Figure shows bar graphs for all variables measured; median telomere length (A), 20th percentile length (B) and percentage of short
telomeres (<3 kbp) (C) in standard condition

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Table 1: Statistical analysis and p-value for median telomere length at different time points for each treatment compared to untreated, paired
samples. Significant differences: *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.
Two-Way ANOVA
Median Telomere Length
Passage 4
Mean 1
Mean 2
Mean Difference
Significance
Ctrl vs. LUT10
6515
6272
243
No
Ctrl vs. LUT5
6515
6340
175
No
Ctrl vs. LUT1
6515
5984
531
Yes (****)
Passage 8
Mean 1
Mean 2
Mean Difference
Significance
Ctrl vs. LUT10
6333
6008
326
No
Ctrl vs. LUT5
6333
6177
157
No
Ctrl vs. LUT1
6333
6071
263
No
Table 2: Statistical analysis and p-value for 20th percentile length at different time points for each treatment compared to untreated, paired
samples. Significant differences: *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.
Two-Way ANOVA
20th percentile telomere length
Passage 4
Mean 1
Mean 2
Mean Difference
Significance
Ctrl vs. LUT10
3393
3160
232
No
Ctrl vs. LUT5
3393
3110
282
Yes (*)
Ctrl vs. LUT1
3393
3356
36
No
Passage 8
Mean 1
Mean 2
Mean Difference
Significance
Ctrl vs. LUT10
3259
2970
289
Yes (*)
Ctrl vs. LUT5
3259
3044
216
No
Ctrl vs. LUT1
3259
2847
412
Yes (***)
Table 3: Statistical analysis and p-value for percentage of telomeres < 3kbp at different time points for each treatment compared to untreated
paired samples. Significant differences: *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.
Two-Way ANOVA
% short telomeres (<3 kbp)
Passage 4
Mean 1
Mean 2
Mean Difference
Significance
Ctrl vs. LUT10
16,9
18,7
-1,8
No
Ctrl vs. LUT5
16,9
19,0
-2,1
No
Ctrl vs. LUT1
16,9
16,9
0,0
No
Passage 8
Mean 1
Mean 2
Mean Difference
Significance
Ctrl vs. LUT10
17,9
20,2
-2,4
Yes (*)
Ctrl vs. LUT5
17,9
19,7
-1,8
No
Ctrl vs. LUT1
17,9
21,3
-3,5
Yes (***)
Telomere Length measurements–Oxidative Stress
Conditions
We wanted to determine the effect of oxidative stress in
telomere length attrition in primary fibroblasts cultures and
evaluate the rate of telomere shortening of the same cultures
in the presence of lutein. This is more/less/equal than the
total shortening observed in the absence of H2O2 (Fig 2 A).
Following treatment under oxidative conditions in the
presence or in the absence of lutein telomere length
variables were determined at 4 and 8 weeks. Data for all
variables determined are presented in the graphs in Figure 3.
Telomere length measurement obtained in oxidative
conditions alone, compared to lutein treated at 4 weeks were
found to be lower in the later (Table 4). Concomitantly an
increase can be observed in the 20th percentile as well as a
decrease in the percentage short telomeres (<3 kbp) (Tables
5 and 6).
After 8 weeks of treatment with Lute-gen® statistical
differences were observed in the median telomere length
values. All Lute-gen® treated groups (10, 5 and 1 µg/ml)
presented a higher telomere length compared to control
(Table 4).

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Fig 3: Figure shows bar graphs for all variables measured; median telomere length (A), 20th percentile length (B) and percentage of short
telomeres (<3 Kbp) (C) in oxidative condition
Table 4. Statistical analysis and p-values for median telomere length at different time points for each treatment compared to the oxidative
paired sample. Significant differences: *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.
Two-Way ANOVA
Median Telomere Length
Passage 4
Mean 1
Mean 2
Mean Difference
Significance
H2O2 vs. LUT10+H2O2
6360
5758
602
Yes (****)
H2O2 vs. LUT5+H2O2
6360
6085
275
No
H2O2 vs. LUT1+H2O2
6360
5980
380
Yes (*)
Passage 8
Mean 1
Mean 2
Mean Difference
Significance
H2O2 vs. LUT10+H2O2
5636
5972
-336
Yes (*)
H2O2 vs. LUT5+H2O2
5636
5980
-344
Yes (*)
H2O2 vs. LUT1+H2O2
5636
6045
-409
Yes (**)
Table 5. Statistical analysis and p-values for 20th percentile length at different time points for each treatment compared to the reference
paired sample. Significant differences: *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.
Two-Way ANOVA
20th Percentile Telomere Length
Passage 4
Mean 1
Mean 2
Mean Difference
Significance
H2O2 vs. LUT10+H2O2
3166
3106
60
No
H2O2 vs. LUT5+H2O2
3166
3359
-193
No
H2O2 vs. LUT1+H2O2
3166
3371
-205
No
Passage 8
Mean 1
Mean 2
Mean Difference
Significance
H2O2 vs. LUT10+H2O2
2998
2964
34
No
H2O2 vs. LUT5+H2O2
2998
2983
14
No
H2O2 vs. LUT1+H2O2
2998
2854
144
No
Table 6. Statistical analysis and p-values for percentage of telomeres < 3kbp at different time points for each treatment compared to the
reference paired sample. Significant differences: *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.
Two-Way ANOVA
% short telomeres (<3 kbp)
Passage 4
Mean 1
Mean 2
Mean Difference
Significance
H2O2 vs. LUT10+H2O2
18,65
19,19
-0,5401
No
H2O2 vs. LUT5+H2O2
18,65
17,01
1,639
No
H2O2 vs. LUT1+H2O2
18,65
16,77
1,882
No
Passage 8
Mean 1
Mean 2
Mean Difference
Significance
H2O2 vs. LUT10+H2O2
20,15
20,14
0,00514
No
H2O2 vs. LUT5+H2O2
20,15
20,1
0,05202
No
H2O2 vs. LUT1+H2O2
20,15
21,15
-1,005
No
Telomere shortening rate
Due to the fact that cell replication is one of the principal
causes of telomere shortening, the telomere length
measurements performed were normalized by the
population doubling levels (cell replication) for each
condition and time point. The telomere shortening rate was
calculated using the following formula; Median telomere
length (initial-final) / Population Doubling.
After 8 weeks under oxidative cell culture conditions (10
µM-H2O2), a decrease in telomere shortening was observed
(Figure 4 B). This decrease was observed in all
concentrations tested and was detected as significant by the
statistical analysis (Table 7).

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Fig 4: The bar chart below displays the average telomere shortening rates of the median telomere length of each condition (control and
treated groups), in standard (A) and oxidative (B) condition
Table 7. Statistical analysis p-value for telomere shortening rate at different time points for each treatment compared to the reference paired
sample. Significant differences: *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001.
Telomere shortening rate – Normal Condition
Passage 4
Mean 1
Mean 2
Mean Difference
Significance
Ctrl vs. LUT10
61
81
-20
No
Ctrl vs. LUT5
61
80
-19
No
Ctrl vs. LUT1
61
100
-38
Yes (****)
Passage 8
Mean 1
Mean 2
Mean Difference
Significance
Ctrl vs. LUT10
41
56
-15
No
Ctrl vs. LUT5
41
55
-14
No
Ctrl vs. LUT1
41
53
-12
No
Telomere shortening rate – Oxidative Condition
Passage 4
Mean 1
Mean 2
Mean Difference
Significance
H2O2 vs. LUT10+H2O2
93
118
-25
Yes (*)
H2O2 vs. LUT5+H2O2
93
93
0
No
H2O2 vs. LUT1+H2O2
93
109
-15
No
Passage 8
Mean 1
Mean 2
Mean Difference
Significance
H2O2 vs. LUT10+H2O2
91
66
25
Yes (*)
H2O2 vs. LUT5+H2O2
91
59
32
Yes (***)
H2O2 vs. LUT1+H2O2
91
63
28
Yes (**)
Discussion & Conclusion
This study aimed to investigate the effect of lutein in
telomere length during cell culture of primary cells using
two general classifications: with and without the pro-oxidant
hydrogen peroxide (H2O2). Different concentrations of
Lutein were included along with positive (H2O2) and
negative (no treatment) controls.
The findings of the study reveal that oxidative stress
decreased proliferation capacity and increased telomere
shortening rate in cultures of human primary fibroblast. For
all treated groups in standard conditions a significant
increase in telomere shortening rate was observed after
LUT1 treatment. For all treated groups in oxidative culture
conditions the observations were that a significant
difference in the telomere shortening rate was identified
between LUT10+H2O2, LUT5+H2O2 and LUT1+H2O2
compared to control (H2O2) after 8 passages.
Numerous studies have shown that oxidative stress is
associated with accelerated telomere shortening and
dysfunction [16, 17]. Due to the antioxidant properties of Lute-
gen both normal and oxidative stress conditions were
included in the study design in order to investigate if the
compound exerts a stronger telomere protective effect under
oxidative stress conditions.
According to the data from the study Lutein has a
significant protective effect on telomere length erosion in
vitro under oxidative stress conditions in 8 weeks.
The evaluation of changes in telomere length including
analysis of the increase of telomeres of critical short length
in cell cultures serves as the demonstration of the protective
effects of lutein (Lute-gen®). Measurement of the rate of
shortening of telomeres along with the accurate
determination of median, 20th percentile and percentage of
telomeres below 3 Kpb, was gathered to examine lutein
impact on telomere dynamics.
A consistent reduction on the attrition rates observed in the
presence of lutein indicates that it has a significant
protective effect on telomere length erosion in vitro under
oxidative stress conditions after eight weeks in culture
during which time X cell divisions took place.
Telomere erosion occurs at a slow pace and can only be
accurately monitored in non-immortalized cells that do not
have constitutive telomerase activity. Although primary
adult cell cultures are suitable to study the effect of culture
conditions and compounds in telomere attrition their
maintenance over long period of time to promote cell
division is also limited. The data obtained following 8
weeks in culture is probably the most representative one
given that the cells had time to undergo more divisions. This
is observed at all three concentrations tested with similar

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90
protective effect for all of them in this long-term experiment
indicating that at concentrations as low as 1 ug/ml lutein can
slow telomere erosion in vitro.
Under normal, non-oxidative conditions the presence of
lutein seems to increase telomere shortening rates after 4
weeks in culture. This effect appears marginally significant
for LUT1. It is unclear the reason why the treatment with
lutein under normal conditions could have accelerated
telomere shortening, one possibility is the side effects of the
excipients present in the treatment during the first weeks of
treatments. Telomere length could be affected by a number
of different factors in addition to proliferation and oxidative
stress, such as activation of nucleases, increased protein
instability of the sheltering-complex (telomere protector),
effect on the expression (transcription / translation) of
different proteins involved in the regulation of telomere
length etc. that were not determined in this study.
The results of the in vitro study performed indicate that
there appears to be evidence that the antioxidant activity of
lutein is beneficial to counter act the effects of oxidative
stress on telomeres in humans. The use of human cell
cultures to obtain proof of concept for the effect on cell
health of compounds such lutein has been demonstrated
with the data presented as they support and provide
evidence that lutein has a direct effect on the proliferation
capacity of fibroblast cells. Further investigation of this
phenomena may lead to understanding of the mechanism of
action of lutein as well as generating information that may
lead to in vivo, clinical studies. The use of such in vitro
modeling is beneficial to investigators attempting to make
initial determinations as to potential effects of a substance
under investigation [8, 9].
The rate of shortening is indeed affected by the treatments
which is something expected. In the case of lutein treated
groups under oxidative stress, we obtained data showing a
slowdown of the shortening rates after eight weeks
indicating a direct protective effect of the compound on
telomeres under these conditions. Telomere attrition during
cell proliferation is mostly caused by the end replication
problem. [10, 11]. The process of telomere shortening is
reduced by the enzyme telomerase, whose purpose it is to
add nucleotides at the end of the DNA molecule.
Telomerase activity is found in some cells (e.g., germ cells
and stem cells), which divide continually and must maintain
telomeres above a critical length in order to perform their
functions [12].
By promoting telomerase activity, it is possible to increase
telomere length and consequently extend the number of
cellular divisions that can take place without incurring in
detrimental chromosomal aberration or telomere fusion.
While this will not make cells immortal, it may extend their
lifespan. We have been able to evaluate a protective effect
on telomeres by comparing cell groups undergoing similar
number of divisions. This theoretically means that the lutein
treatment has a protective effect on telomeres, but this
statement needs to be confirmed by taking into account the
division of the cells during the expansion period. In the case
that Lute-gen would halt the division of the cells, the cells in
the control-untreated group would expectedly undergo
several more divisions in comparison to the treated groups.
In that case telomere length in the treated groups would be
higher not because of the compound’s protective effect on
telomeres, but simply because it prevented the cells from
dividing sufficient times. For this reason, the data was
normalized taking into account the cell divisions in each
group and the telomere shortening rate was thus calculated.
It is very interesting that the protective effect under
oxidative stress conditions after 8 weeks of treatment, is
indeed observed after the correction factor is included in
analysis of the study. We can therefore conclude that in
vitro we are observing a protective effect under oxidative
stress conditions and not simply a decline in the
proliferation rate [13].
However, whether the administration of lutein reduces the
level of ROS in other cell types and has a positive effect on
healthy aging needs to be elucidated which may be beyond
the scope of this work.
References
1. Estrada JC, Albo C, Bengur.a A, Dopazo A, L.pez-
Romero P, Carrera-Quintanar L. et al. Culture of
human mesenchymal stem cells at low oxygen tension
improves growth and genetic stability by activating
glycolysis. Cell Death and Differentiation. 2012;
02(19):743-755.
2. Spence AP. Biology of Human Aging, 2nd ed. Prentice
Hall, Upper Saddle River, NJ, 1999.
3. Bryan TM, Reddel RR. Telomerase, Immortality and
Cancer. Today’s Life Science. 1996; 8:26-28.
4. Hayflick L. Theories of biological aging: Programmed
Aging. Experimental Gerontology. 1985; 20:145-159.
5. Zhu H, Guo D, Li K, Pedersen-White J, Stallmann-
Jorgensen IS. Increased telomerase activity and vitamin
D supplementation in overweight African Americans.
Int J Obes (Lond). 2012; 36:805-9.
6. Canela A, Vera E, Klatt P, Blasco MA. High-
throughput telomere length quantification by FISH and
its application to human population studies. Proc Natl
Acad Sci USA. 2007; 104(13):5300-5.
7. Kimura M1, Stone RC, Hunt SC, Skurnick J, Lu X, Cao
X. et al. Measurement of telomere length by the
Southern blot analysis of terminal restriction fragment
lengths. Nature Protocols. 2010; 5(9):1596-607.
8. Richards JB, Valdes AM, Gardner JP, Paximadas D,
Kimura M. Higher serum vitamin D concentrations are
associated with longer leukocyte telomere length in
women. Am J Clin Nutr. 2007; 86:1420-5.
9. Chan R, Woo J, Suen E, Leung J, Tang N. Chinese tea
consumption is associated with longer telomere length
in elderly Chinese men. Br J Nutr. 2010; 103:107-13.
10. Wright WE, Tesmer VM, Huffman KE, Levene SD,
Shay JW. Normal human chromosomes have long G-
rich telomeric overhangs at one end. Genes Dev. 1997;
11:2801-2809.
11. Wright WE, Tesmer VM, Liao ML, Shay JW. Normal
human telomeres are not late replicating. Exp Cell
Res. 1999; 251:492-499.
12. Sen A, Marsche G, Freudenberger P, Schallert M,
Toeglhofer AM. Association between higher plasma
lutein, zeaxanthin, and vitamin C concentrations and
longer telomere length: results of the Austrian Stroke
Prevention Study. J Am Geriatr Soc. 2014; 62:222-9.
13. Xu Q, Parks CG, DeRoo LA, Cawthon RM, Sandler
DP. Multivitamin use and telomere length in women.
Am J Clin Nutr. 2009; 89:1857-63.
14. O’Callaghan NJ, Fenech M. A quantitative PCR
method for measuring absolute telomere length. Biol
Proced Online. 2011; 13:3.

International Journal of Biology Research
91
15. Proctor CJ, Kirkwood TB. Modelling telomere
shortening and the role of oxidative stress. Mech
Ageing Dev. 2002; 123(4):351-363.
16. Kawanishi S, Oikawa S. Mechanism of telomere
shortening by oxidative stress. Ann N Y Acad Sci.
2004; 1019:278-84.
17. Tchirkov A, Lansdorp PM. Role of oxidative stress in
telomere shortening in cultured fibroblasts from normal
individuals and patients with ataxia-telangiectasia. Hum
Mol Genet. 2003; 12(3):227-32.