ASSESSMENT OF GENOTOXICITY OF SODIUM ARSENITE IN CULTURED HUMAN LYMPHOCYTES
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 effect of different concentrations of arsenic on human chromosomes 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 chromosomal anomalies.The leukocyte 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 sources. Arsenic is a well known 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 routes 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 with oxygen and sodium. Sodium Arsenite (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 sequential process in which the pentavalent 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 biomarker 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, neuronal and metabolic disorders have been seen. Acute exposure is seen to result in vomiting, nausea, abdominal pain, neuropathy 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 genotoxic 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 are washed with alcohol prior to planting .To a fresh tube, 5ml of Hikaryos RPMI 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 standing 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 72nd hour of incubation 60 microliters of colchicine is added to each tube and the tubes are kept for 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 with 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 rpm 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 white.
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 metaphase
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 are 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.
right5080 5µM SODIUM ARSENITE
00 5µM SODIUM ARSENITE
21145505080 1µM SODIUM ARSENITE
00 1µM SODIUM ARSENITE
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 treated 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.
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 aches, 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 and 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 infertility 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 present study on human leukocyte culture has verified the genotoxic effect of arsenic.
1 Souza, A.C.F., Marchesi, S.C., Ferraz, R.P., Lima, G.D.D.A., Oliveira, J.A.D. and Machado-Neves, M., 2016. Effects of sodium arsenate and arsenite on male reproductive functions in Wistar rats. Journal of Toxicology and Environmental Health, Part A, 79(6), pp.274-286.
2 Balasubramanian, J. and Kumar, A., 2013. Effect of sodium arsenite on liver function related enzymes of cat fish Heteropneustes fossilis and its chelation by zeolite. Ecotoxicology and Environmental Contamination, 8(2), pp.53-58.
3 Gradecka, D.O.B.R.O.S.?.A.W.A., Palus, J.A.D.W.I.G.A. and Wasowicz, W., 2001. Selected mechanisms of genotoxic effects of inorganic arsenic compounds. International journal of occupational medicine and environmental health, 14(4), pp.317-328.
4 Sarker, R.S.J., Ahsan, N. and Akhand, A.A., 2012. Sodium arsenite induced systemic organ damage and changes in various blood parameters in mice. Dhaka University Journal of Pharmaceutical Sciences, 11(2), pp.169-172.
5 Hungerford, D.A., 1965. Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCl. Stain technology, 40(6), pp.333-338.
6 Helleday, T., Nilsson, R. and Jenssen, D., 2000. Arsenic III and heavy metal ions induce intrachromosomal homologous recombination in the hprt gene of V79 Chinese hamster cells. Environmental and molecular mutagenesis, 35(2), pp.114-122.
7 Nordenson, I., Beckman, G., Beckman, L. and Nordström, S., 1978. Occupational and environmental risks in and around a smelter in northern Sweden: II. Chromosomal aberrations in workers exposed to arsenic. Hereditas, 88(1), pp.47-50.
8 Nordenson, I., Salmonsson, S., Brun, E. and Beckman, G., 1979. Chromosome aberrations in psoriatic patients treated with arsenic. Human genetics, 48(1), pp.1-6.