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Assessment of Basal DNA Damage in Normal and Healthy

Young Individuals by COMET Assay A K DIXIT* and ARUN N.

Dept of Plant Biology and Biotechnology

KM Centre for PG studies, Pondicherry 605008 India

*akd7@in.com Abstract

The present study has designed to assess the levels of DNA damage in healthy

individual. To compare the levels of DNA damage among the individuals of

similar age group with gender, food habits and lifestyle as well as the

environmental factors. Life style and personal habits can produce a low level

DNA damage and Comet formation in normal healthy individuals are due to this

primary DNA lesions. These primary DNA damage may leads to biologically

relevant chromosome or gene mutations. This study was undertaken with the

aim of assessing the status of DNA damage in a normal healthy population of

Pondicherry by using COMET assay. The 60 male and 30 female volunteers in

this study belonged to the smoking, non-smoking, vegetarian and non-

vegetarian, non-alcoholic and alcoholic categories and aged between 25 and 35

years. Data from the present study showed a marked difference in the Comet

formation of the individuals when compare their area of residents i.e. in terms of

exposure to environmental pollutants and eating habits. However factors like

level of exercise, infection, occupational exposure i.e. exposure of the

individuals to chemicals and other mutagens should be recorded and studied to

make conclusion about the basal level of DNA damage in healthy individuals.

Keywords:

Basal DNA damage,

Healthy individual,

Comet assay

1. INTRODUCTION

DNA is a relatively fragile molecule which

can be easily damaged in vivo by a number

of different elements. It is a well known that

there is a certain level of basal DNA damage

even in normal healthy individuals due to

environmental pollutants/mutagens exposure

as well as from personal habits of the

individual. For example, a local pH change

may cause the loss of constituent purine

bases; chemical agents from the

environment may modify one or more bases;

and X-rays or ultraviolet (UV) radiation

(long term exposure to sunlight) may bring

about a chemical change in a base. Such

changes in DNA can be the result of normal

metabolism and this kind of damage can be

increased by malnutrition, various toxins, or

a disease state.

Comet assay has become a principle tool in

human bio monitoring studies. In the last

few decades the assay has got wide

acceptance due to its simplicity, sensitivity,

versatility, speed, and economy. The assay

is less expensive when compare to other

biomonitering assays. Samples can be

prepared and evaluated very quickly by

visual scoring [1] or by using image analysis

systems. Data can be generated fast and

efficiently. The comet assay has widespread

applications in genotoxicity testing in vitro

and in vivo, human biomonitoring or

ecotoxicology research [2] [3].

Ostling and Johannson developed a

microelectrophoretic technique under

neutral conditions to study DNA damage in

individual cells after gamma-irradiation and

proposed that strand breaks would enable

DNA loops to stretch out upon

electrophoresis and form the comet tail [4].

Singh et al. introduced the alkaline version

of the comet assay which was able to detect

alkaline-labile sites, crosslink and transient

DNA strand breaks arising due to DNA

repair processes in addition to DNA breaks

detection [5] . Relaxation of loops was also

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proposed to be the primary underlying basis

for comet formation under alkaline

conditions [6] [7] demonstrates that the

comet tail under neutral conditions consists

of relaxed loops, whereas comet tails under

alkaline conditions consist of DNA

fragments. Singh and Stephens proposed

that some single strand breaks might reflect

DNA-protein complexes [8]. They

suggested that residual protein in microgel

slides should be removed and, thereby,

increases the sensitivity of the assay. It has

been proposed that Cytotoxic effects can be

detected and distinguished from genotoxic

effects and, therefore, should have no

confounding effects on results [9] [10] [11].

Cytotoxicity produce cell death and dead

cells show specific kinds of comets called

'clouds' or 'hedgehogs'.

The comet assay has also shown high

sensitivity for detecting carcinogenicity and

due to this it is highly useful in predicting

the risk for Cancer, so it is a very useful tool

for

human biomonitoring studies. Several

versions of the comet assay are in use and

can be done in alkaline or neutral conditions,

the neutral method for the detection of DNA

double-strand

breaks, and the alkaline

method, which detects DNA single-strand

breaks and alkali-labile lesions. Glei et.al.

studied the susceptibility and potential

exposure risks or risk reductions of diet and

lifestyle in various tissues by comet assay

and suggested that food habits and lifestyle

of the individuals had a role in

tumerogenesis [12].

2. MATERIALS AND METHODS

This study was undertaken with the aim of

assessing the status of DNA damage in a

normal healthy population of Pondicherry

by using COMET assy. All individuals

answered a questionnaire that assessed their

health status, dietary habits, lifestyle, and

medication. These healthy selected

volunteers signed a letter of consent before

participation in this study. The 60 male and

30 female volunteers in this study belonged

to the smoking, non-smoking, vegetarian

and non-vegetarian, non-alcoholic and

alcoholic categories and aged between 25

and 35 years. The details of all the healthy

subjects are mention in Table.-1. The reason

for selection of particular age group for the

study was they were apart from diabetes;

leukaemia, cancer and they may have less

DNA damage.

The single-cell gel electrophoresis( SCGE)

or Comet assay is a rapid non-invasive

technique which measures DNA damage in

individual cells and useful in biomonitoring

studies to determine the level of DNA

damage resulting from exposure to

occupational and environmental mutagens

and also from lifestyle of individuals. This

simple and sensitive technique, first

introduced by [13] to visualize the relative

extent of single stranded DNA to double

stranded DNA. The cells were embedded in

an agarose layer, exposed to alkaline

condition (pH-12) thus allow the unwinding

of the double stranded DNA. When stained

with Acridine orange the single stranded

DNA emitted red signals and double strands

emitted green signals. The amount of single

stranded DNA was calculated from the

intensity of red signals relative to green

signals. In 1984 this technique was

modified by including electrophoresis

instead of alkaline treatment and there by

stretching of damaged DNA towards the

anode for direct visualization of damaged

DNA in individual cell [5]. The alkaline

version of this method was introduced by

[14] by including alkaline condition (pH

>12) during the electrophoresis. Comet

assay has become a major tool in the fields

like toxicology, apoptosis, DNA repair,

ageing, cell cycle analysis and free radical

biology due to its sensitivity to express the

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single/double-strand breaks, DNA-drug

crosslinking, DNA-protein or DNA-DNA

crosslinking and oxidative DNA base

damage.

Generally 03 ml of Heparinised blood has

collected from all the individuals and the

lymphocytes were separated using

Histopaque® 1077 [15].

2.1. Lymphocyte separation:

From 02 ml of whole blood white cells are

separated using Histopaque 1077®

. In brief 2

ml of Histopaque has transferred to a 15 ml

conical centrifuge tube and 02 ml of whole

blood (mixed with Sodium heparin) layered

over it without disturbing it. Centrifuged the

tube for 30 mins at 400g. After the

centrifugation the middle layer containing the

Lymphocytes are carefully pipetted out and

transferred to a microcentifuge tube

containing PBS (pH 7.2). Mix the cells in

PBS with help of pipette. Cells were diluted

in PBS to a concentration of 4x104 cells/m.

2.2. Encapsulation:

Slides are prepared in an air conditioned

room adjusted at 25ºC in brief onto clean,

dry, plain glass slides about 150µl of hot, 1%

NMPA is dropped and smeared in one

direction with the help of another plain slide

inclined at about 45ºC. Once NMPA is set,

75µl LMPA (37%C) mixed with 20µl of

lymphocytes (from the above preparation), is

added onto the NMPA layer. The cover slip

is replaced and the gel is again allowed to set

at 4%C for 5-10mins. After 10 mins the

cover slip is gently removed and 75µl of

LMPA is added onto the second agarose

layer. The cover slip is replaced and agarose

is allowed to set again at 4ºC for 5-10mins.

Three slides were made for each sample. The

agarose forms a matrix of carbohydrate fibres

that encapsulate the cells, anchoring them in

place. The agarose is considered to be

osmotic-neutral, therefore solutions can

penetrate the gel and affect the cells without

cells shifting position.

2.3. Lysis:

The cover slip are removed again and the

slides are gently submersed into cold Lysing

solution and refrigerated for a minimum

12hrs at 4ºC. The lysis solution often used in

the comet assay consists of a highly

concentrated aqueous salt (often, common

table salt can be used) and a detergent (such

as Triton X-100 or sarcosinate). The aqueous

salt disrupts proteins and their bonding

patterns within the cell as well as disrupting

the RNA content of the cell. The detergent

dissolves the cellular membranes. Through

the action of the lysis solution the cells are

destroyed. All proteins, RNA, membranes

and cytoplasmic and nucleoplasmic

constituents are disrupted and diffuse into the

agarose matrix. Only the DNA of the cell

remains, and unravels to fill the cavity in the

agarose that the whole cell formerly filled.

This structure is called nucleoid (a general

term for a structure in which DNA is

concentrated).

2.4. Electrophoresis:

After lysis of the cells (typically 1 to 2 hours

at 4°C) the slides are washed in distilled

water to remove all salts and immersed in a

second solution - an electrophoresis solution.

Again this solution can have its pH adjusted

depending upon the type of damage that is

being investigated.

The slides are left for ~30 minutes in the

electrophoresis solution prior to an electric

field being applied. In alkaline conditions the

DNA double helix is denatured and the

nucleoid becomes single stranded.

An electric field is applied (typically 1 V/cm)

for ~20 minutes.

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The slides are then neutralised to pH 7, with

neutralizing buffer (0.4M Tris pH 7.4) to sit

on the slide for 10 mins the buffer is drained;

the process is repeated two more times. After

neutralized with Tris Buffer the slides are

dried for about an hour at room temperature.

These slides are placed in fixing solution (A)

for 10min and again washed several times

with double distilled water. The slides are

then allowed to dry at 37ºC for a maximum

of overnight. The slides are placed in a tray

and fresh silver staining solution is poured

gently over the slides and shaken very gently

to ensure uniform staining of the slides, until

a greyish colour develop on the slides. The

slides are then transferred to a jar containing

the stopping solution for about 5min or until

a yellowish brown colour develops. The

slides are then washed again with water and

allowed to dry in inclined position at room

temperature. Silver stained slides are

observed under a Trinocular research

Microscope (Nikon Opti Phot) using a 40X

objective. DNA damage is estimated as

length of the comet tail which is measured

using an Ocular Micrometer fitted in the

eyepiece of the microscope.

3. RESULTS

The data revealed significant differences in

the extent of DNA damage in the smokers

versus non-smokers as well as between the

vegetarians and non-vegetarians. A

significant difference was also observed

amongst the different groups of smokers

depending on the extent of smoking. This

preliminary study has, for the first time,

revealed differences in the extent of DNA

damage in the normal young population of

Pondicherry depending on their eating and

smoking habits(Fig-1&2).

3.1. Effect of Age on Comet Assay

response

In the present study the statistical analyses

the effect of the age of the individual in the

comet assay has not shown any significant

changes but the graphical representation of

the comet tails is showing a gradual increase

in comet length from the lower age group to

the higher age group. There are conflicting

results among the previous studies regarding

the age related changes in comet parameters.

Among the earlier reports only few studies on

healthy individuals demonstrated changes in

the comet parameters according to the age

difference (16, 17,18) major number of

studies (8, 9, 19, 20, 21, 22 ) in smaller as

well as larger population did not detect an

age-related effect on the level of

DNA

damage. The present study suggest that the

age of the individuals who participated for

the study have only a little effect on the

mean basal level of DNA damage (Fig-3&4).

As part of one’s life style and affect of

environmental pollutants, free radicals and

other reactive oxygen or nitrogen species are

constantly generated in vivo and can cause

oxidative damage to DNA which leads to

many diseases, including cancer [19]. Earlier

studies [6-12] showed that factors such as

gender, age and diet influence Comet assay

responses in lymphocytes.

In the present study the deference in the

mean Comet Length is highly significant in

the males of the Urban population (P value =

0.0002) when compare to rural population.

But in females the values are not

significant.(Table-2&3 and Fig-5&6)

In the present study the mean value of Comet Length among both sexes are of no significant (P value = 0.1243). There are conflicting reports in the earlier studies about DNA damage consist of individuals of both sexes showed that men had more DNA damage than women (23, 24). But in a few

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reports [15] women had more basal DNA damage than men.

4. DISCUSSION

Life style and personal habits can produce a low level DNA damage and Comet formation in normal healthy individuals are due to primary DNA lesions. These primary

DNA damage may leads to biologically relevant chromosome or gene mutations. However factors like level of exercise, infection, and diet, occupational exposure i.e. exposure of the individuals to chemicals and other mutagens should be recorded and studied to make conclusion about the basal level of DNA damage in healthy individuals.

FIG: 1

FIG: 2

FIG: 3

Gender deference in Comet Length

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Male & Female subjects

Mea

n C

omet

Len

gth

Age vs Comet Length in the Study Group

0

0.02

0.04

0.06

0.08

0.1

0.12

Min Age Group Max Age Group

Lower & Higher Group of Ages

Mea

n Co

met

Len

gth

Mean Comet Length Males Mean Comet Length Females

A k Dixit etal ,J Biosci Tech, Vol 2 (2),2011,247-257

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Age deference & Comet Length in male subjects

FIG: 4

Age deference & Comet Length in female subjects

Age Below 29 Age above 30

Mean Comet

Length

Males 0.09283 0.107

Females 0.07956652 0.09398926

FIG: 5

25

2627 28

30

3134

35

0

0.02

0.04

0.06

0.08

0.1

0.12

25 26 27 28 30 31 34 35

26

2829

30

31

34

35

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

26 28 29 30 31 34 35

A k Dixit etal ,J Biosci Tech, Vol 2 (2),2011,247-257

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Deference in area of residence & Comet Length in male subjects

FIG: 6

Deference in area of residence & Comet Length in female subjects

Table - 1:

Subject details

Subject Sample

size

Marital

status Food

Drinking

Residence Alcoholic

smoking

M UM NV PV Cof. Tea R U A NA

Male 60 33 27 56 04 13 47 22 38 49 11 41

Female 30 24 06 21 09 03 27 17 13 09 21 03

Total 90 57 33 77 13 16 74 39 51 58 32 44

M-Married, UM-Unmarried, NV-Non vegetarian, PV-Pure vegetarian,

Cof. - Coffee, R-Rural, U-Urban, A—Alcoholic, NA-Non alcoholic.

Table - 2

Rural vs. Urban in Male subjects

0.01

0.1

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Rural Urban

Female subjects -Rural vs Urban

0.01

0.1

1

1 2 3 4 5 6

Female subjects

Mea

n c

om

et le

ng

th

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Parameter: Rural Urban

Men Comet Length 0.0894646154 0.1051828571

Standard deviation (SD) 0.007245 0.006783

Std. error of mean(SEM) 0.002009 0.002564

Minimum 0.07960 0.09688

Median (50th percentile) 0.08772 0.1046

Maximum 0.1020 0.1144

Unpaired t test two-tailed P value = 0.0002(very significant).

Table – 3

Rural vs. Urban in Female subjects

Parameter: Rural Urban

Mean Comet Length: 0.08079 0.09576

Std deviation: 0.01271 0.02317

Std error: 0.005189 0.01158

Minimum: 0.06296 0.07280

Maximum: 0.09599 0.1270

Median: 0.08214 0.09164

Two-tailed P value = 0.2189 (not significant)

Nakayama et al. reported DNA strand break

in smokers due to the effect of electrophillic

substances in tobacco [25]. Jayakumar and

Sasikala reported cigarette smoking habitual

has a synergistic effect on including DNA

damage among the jewellery workers are

occupationally exposed to nitric oxide [26].

The present findings highlight the

importance of using comet assay to detect

DNA damage and genotoxic effect of

various confounding factors, since this

information provides an increased degree of

identification for the positive response.

In order to study comet-effects in more

detail, [6] proposed to use lesion-specific

enzymes also in human monitoring studies.

Physical exercise-induced DNA damage due

to oxidative stress has studied extensively. A

reduction in DNA damage was noticed after

exercise by the supplementation of vitamins

in the volunteers [27][28][29][30][31].

DNA strand breaks are believed to arise

naturally as part of a developmental

program. Vatolin et al. [32] used comet

assay and studied the origin of single strand

breaks as part of the development & cell

division. They found a dramatic increase in

SSB during the first 2-4 mitosis after the

beginning of differentiation of embryonic

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stem cells in vitro. The increased levels

corresponded with increased levels of

SCGE. It was concluded that about half of

the chromatin of the cells had been nicked at

those stages, which, however, did not result

in a phenotypic manifestation. In another

similar study using comet assay to measure

DNA effects in embryos from 7.5 day old

mice and also found high levels of DNA

breaks in the cells [25].These results showed

though, that there are comets which do not

have a biological significance in terms of

mutagenesis.

Some investigators describe increased

comets in leukocytes of smokers [25] or

show decreasing comets when volunteers

quit smoking [27] others do not find a

difference in smokers versus non-smokers

[6][24]. This may, in part, be attributed to

the examination of cells such as leukocytes,

which are not directly exposed to cigarette

smoke. By using exfoliated buccal cells,

[33] demonstrated clearly increased DNA

damage in smokers. Radak et al.

[34]

showed an increased repair of oxidative

DNA damage directly after exercise.

5. CONCLUSION

In conclusion bio-monitoring studies of

healthy young populations are rather

specific because each population has a

different life style factors in different areas

under different climatic and environmental

conditions and are exposed to

indistinguishable of mutagen. Since DNA

damage is an important step in events

leading from carcinogen exposure to cancer

disease, our study represents an important

contribution to the correct evaluation of the

potential health risk associated with

exposure. The comet assay detects primary

DNA lesions (DNA damage) such as DNA

strand breaks. These lesions may not be of

much relevance since they may be repaired

error-free or represent transient repair sites.

We conclude that environmental conditions

play a role in the accumulation of DNA

damage. Our study investigated the

relationship between age and environmental

conditions which contribute a great deal in

setting a higher degree of basal DNA

damage. An antioxidant-rich environment

may help to provide optimal conditions for

maintaining DNA and prevent DNA

damage. The use of different antioxidants

and scavengers in maintaining DNA and

prevent DNA damage is in progress.

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