ASSESSMENT OF GENOTOXICITY OF SODIUM ARSENITE IN CULTURED HUMAN
Atreyee Borthakur,Anupam Dutta,Monica Bhati and V.G.Abilash
School of Biosciences and Technology, Vellore Institute of Technology
Long term exposure to arsenic due to groundwater contamination causes a great threat to human
health conditions. Arsenic being a potential carcinogen is known to cause genetic damage
ultimately resulting in chronic poisoning, skin lesion and cancer. The ef fect of different
concentrations of arsenic on human chromosome s were investigated. Human leukocyte culturing
was carried out to evaluate the chromosome number and damage due to arsenic. The present study
showed that exposure to arsenicals result in chromo somal anomalies. The leucocyte culture
analysis showed the genotoxic effect of arsenic on human chromosomes.
KEYWORDS: Sodium Arsenite, Genotoxicity, Chromosome, Arsenicals
In day to day life human beings are exposed to several toxicants of which arsenic is the potent one.
Arsenic exposure is an important problem around the globe with about 100 million individuals at
risk through various natural and anthropogenic source s. Arsenic is a well know n carcinogen and
teratogen and can be toxic when present in higher amount. It is a heavy metal found in soil, air or
water in both organic and inorganic forms 1 . Degradation of rocks and ores causes gradual
leaching of arsenic into ground water , making contaminated water the major source of arsenic
exposure 1 . Other r outes include mining activities, presence in pesticides or agriculture runoff as
well as industrial effluents, further adding to contamination of drinking water 2. Inorganic form
of arsenic may be found in combined state wit h oxygen and sodium. Sodium A rsenite (NaAsO2)
is an inorganic compound and one of the most toxic forms that inhibits biochemical events by
reacting with endogenous thiol groups 1 . The metabolism of inorganic arsenic is however known
to occur by a sequent ial process in which the pentava lent arsenic Arsinate , As ( V) is reduced to
trivalent arsenic Arsenite, As ( lll) followed by oxidative methylation to pentavalent arsenic 3 .
The toxicity of arsenic depends upon its chemical form and oxidation state . Both trivalent and
pentavalent forms are basically absorbed through GI tract. Blood arsenic as well as concentration
of arsenic in nails, urine or hair may serve as an effective biomar ker in case of acute or high level
chronic exposure. Chronic exposure results in accumulation of arsenic in heart, lungs, kidney ,
liver, spleen and gastrointestinal tract (GI) , however the most significant effect being hepatic
damage 4. Reports of skin, lung, kidney cancer and cardiovascular, neuron al and metabolic
disorders have been seen. Acute exposure is seen to result in vomiting, nausea, abdominal pai n,
neur opathy and encephalopathy. Some studies have suggested an association of arsenic
contamination to infertility and reproductive toxicity in males affecting sperm count and motility
1 . The genoto xic effect of inorganic arsenic can cause deletion mutation in human cells leading
to chromosomal aberrations such as chromatid gaps, fragmentation as well as micronucleus
formation 3 . Studies have also suggested mechanism of arsenic affecting DNA including
oxidative stress and inhibition of DNA repair mechanism 3. These abnormalities can lead to
malignancies and these damages can be measured using karyotyping and CBMN assay analysis.
MATERIALS AND METHODS
ANALYSIS OF HUMAN LEUKOCYTE CULTURE –
The protocol is adapted from Hungerford 1965 5.
The UV light of the Laminar Air Flow (LAF) is switched on for 30 minutes prior to the planting
experiment. After 30 minutes, the UV light is switched off and the air blower of the LAF is started.
The basement of the LAF is cleaned with 70% alcohol to remove bacterial contamination, if any.
Now all the requirements are placed inside the laminar air flow and a spirit lamp is lighted. Our
hands ar e washed with alcohol prior to planting .To a fresh tube, 5ml of Hikary os RPM I ready mix
media and 0.5ml of heparinized blood (50 drops) is added. The contents of the tube are mixed
gently by shaking. The tubes are placed in incubator for 72 hours in stand ing position and CO2 is
released after every 24 hours by slightly rotating the screw cap of the tube.
HARVESTING OF HUMAN LEUKOCYTE CULTURE –
At the end of 72 nd hour of incubation 60 microliters of colchicine is added to each tube and the
tube s are kept f or 20 minutes incubation in 37 °C incubator. After 20 minutes of incubation, the
content is centrifuged at 1500 rpm for 10 minutes. After centrifugation, the supernatant is removed
carefully and 6ml of hypotonic solution is added. The contents are mixed wit h Pasteur pipette and
incubated at 37 °C for 6 minutes. After incubation, the contents of the tube are taken out and
centrifuged at 2000rpm for 5 minutes. After centrifugation the supernatant is discarded and 6ml of
Carnoy’s fixative is added and mixed vigorously (after adding fixative the contents are mixed
immediately by using Pasteur pipette to avoid formation of lumps). After fixation, the tubes are
kept at room temperature for 1 hour. The content is centrifuged at 1500 r pm for 10 minutes. After
discarding the supernatant, 6ml of carnoy’s fixative is added and mixed vigorously. The content
is centrifuged at 1500rpm for 10 minutes. The supernatant is discarded and the washing step is
continued until the pellete becomes whit e.
BASIC PREPARATION OF HUMAN LEUKOCYTE CULTURE –
The slides are first washed in chronic acid and kept in distilled water inside deep freezer in chilled
condition. Later during the procedure the slides are taken out and held in standing position and 3-
4 drops of pellet mixed with 1ml of fixative is dropped over the slides (chilled) and kept in the
oven until dry. The slides are then viewed under 10X power of microscope to confirm for the
STAINING OF HUMAN CHROMOSOME –
The working stain is prepared and poured in a clean coplin jar. Another clean coplin jar is taken
and 50ml of double distilled water (destaining solution) is poured in it. The slides are placed in a
coplin jar containing Giemsa stain for 4 minutes. For destaining, the slides a re placed in a coplin
jar containing double distilled water for 1 minute. The slides are air dried thoroughly and viewed
under microscope for stained chromosome. Mounting with a cover slide is done if desired.
Around 10 metaphases in each culture tube were analysed. The chromosomes in the 1µM sodium
arsenite culture showed acentric chromosomes, presence of chromatid break and chromosomal
lesions. Whereas, the culture tubes treat ed with 5µM sodium arsenite showed chromosome break
along with acentric chromosomes, chromosomal lesions and chromatid breaks. On the whole the
5µM treated chromosomes were comparatively reduced or shortened in size .
CONTROLLED 1µM Sodium arsenite 5µM Sodium arsenite
Sodium arsenite may be absorbed through the epidermis, inhaled or may reach the blood stream
by consumption of contaminated drinking water. Mere contact with the skin may result in rashes,
burns or loss of pigment while consumption may lead to stomach ache s, vomiting, convulsions,
diarrhoea, headaches etc. Chronic consumption has also been linked to keratosis,
hyperpigmentation, renal dysfunction, peripheral neuropathy, peripheral vascular diseases and
other neurological disorders . 2 Long term exposure an d uptake of considerable amounts of
inorganic arsenic may increase the chances of developing cancers, especially cancers of the liver,
skin, lung or lymphatic cancer. Studies also suggest an association of arsenic exposure to
reproductive defects like infe rtility in men. 1
A study performed on male mice showed that daily consumption of arsenic through drinking water
resulted in damage to the reproductive system 1. Helleday et al 2000 showed enhanced DNA
damage, sister chromatid exchange and chromosomal abnormalities due to arsenic in human
leukocytes through in vitro studies 6. Inhalation and oral exposure to arsenic have shown
chromosomal damage in human 7 8.
Arsenic exposure has a damaging effect even in low concentrations. These include chromosome
break, chromatid break, shortening of telomere and chromosomal lesions. The long term exposure
to arsenicals may finally lead to chromosome fragmentation. The presen t study on human
leukocyte culture has verified the genotoxic effect of arsenic.
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