MN & CA: Biomarkers for Genotoxicity
Across the Globe, it is known from ancient time, mineral fibres having great deal of importance for its use in the manufacturing of different kind of mineral based-products. Asbestos is one of the fibrous mineral silicates, which accounts more than 3,000 of its uses all over the World (Ramanathan and Subramanian, 2001). In India, there are about 13 large and 673 small-scale asbestos based industrial units. Along with this, it is also estimated that asbestos industry directly gives employment to 6,000 workers, and another 1,00,000 peoples indirectly dependent on its usages. The epidemiological and biochemical changes induced by asbestos in relation to their occupational health hazards have been well documented (Mossman et al., 1990). Asbestos has been known for its carcinogenic and co-carcinogenic property (Mossman et al., 1990). Asbestos related diseases take a long latency period of 10 to 40 years before appearance of the obvious symptoms. The risks of carcinogenic and co-carcinogenic effects from asbestos continue owing to the persistence of the fibres from mining, milling, manufacturing and its use in building materials and other products and it is well known that the human population is exposed to complexes of mixture of different pollutants via occupationally, domestically and or, environmentally.
For this reason, epidemiological and mechanistic research on the toxic effects of asbestos and minerals fibres is still needed.
Exposure to asbestos causes various lesions ranging from simple non-malignant inflammatory reactions, pleural thickening and asbestosis to malignant mesothelioma and bronchiogenic carcinoma (Mossman et al., 1990). As mentioned earlier, asbestos fibres are well known environmental carcinogen, however, the underlying mechanisms of their action have still not clearly been identified. To determine the genotoxic risk associated to environmental and occupational exposure of asbestos; micronuclei (MN) and Chromosomal aberrations (CA) analysis techniques are being used.
Micronuclei (MN) and Chromosomal aberration (CA) analysis techniques across the Globe are being emerging tool as a biomarker for the genotoxic risk to asbestos as earlier mentioned. Studies conducted In Vitro and experimental animals have shown that asbestos is a genotoxic carcinogen, causing chromosomal breaks and deletions (Okayasu et al., 1999). Previous studies done in our laboratory has reported that the induction of MN and CA in lymphocytes of workers occupationally exposed to asbestos (Rahman et al., 2000).
The studies of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis and these techniques have been emerged as one of the preferred methods for assessing chromosome damage.
Studies in lymphocytes culture system are a valuable tool by which toxicant potential of xenobiotic can be assessed at genetic level. Blood is the most frequently used tissue specimen for cytogenetic diagnosis for a variety of clinical cases because peripheral blood can be easily obtains from subjects of all age groups. For chromosomal aberration and micronuclei analysis methods of Moorhead et al., 1960 and Fenech, 1993 are being used.
Accordingly, lymphocytes from the peripheral blood are stimulated by the Mitogen, phytohaemagglutinin (PHA) to divide in culture and a large no. of metaphases can be seen 72 hours after setting up of culture. The culture medium contains 5 ml RPMI-1640, 15% fetal/ borne calf serum, 1% L-glutamine, 1% Sod. Heparin (100 units/ml), 1% penicillin streptomycin containing 10,000 units/ml penicillin, 10000 mcg/ml speptomycin, 2% PHA. Incubation continues for 72 hours. Colchicine (10 mcg/ml) is added 1 hr prior to harvest cells at metaphase stage. The cell suspension is kept in prewarmed hypotonic (0.075 M KCl) solution to swell the metaphase then the cells are fixed with Carnoy’s fixative to prepare slides for microscopic observation of chromosomal aberrations.
Any kind of damage or alterations in the normal structure of a chromosome which are visible under microscope and are the outcome of the damage caused to the DNA, are called as chromosomal aberration, such as chromosomal and or chromatid gap and break, acentric, polycentric, ring, triradial chromosomes etc.
The structural aberrations observed in metaphase cells are basically of two types: Chromatid gap and Chromatid break.
Chromatid gap is an unstained region in the chrosomatid and on very careful examination, fine threads can sometimes be seen running across the nonstaining region.
Chromatid break is similar to chromatid gap but the terminal part of the chromatid has been so displaced as to indicate that is no longer attached in any way to the proximal part of the chromosome.
The study of MN as a measure of chromosome damage in peripheral blood lymphocytes (PBL) was first proposed by Courtyman and Heddle (1976) and sub-sequently improved with the development of cytokinesis-block micronucleus (CBMN) method (Fenech and Morley, 1985a,b), which allowed micronuclei to be scored specifically in cells that had completed nuclear division. Chromosomes are complexes of nucleic acids and proteins; DNA is ofcourse, the essential components of the chromosome, which may be regarded simply as device for carrying the genetic information from a parent cell to its daughters.
MN in peripheral blood lymphocytes is well-established cytogenetic techniques that have been used extensively in human bio monitoring for assessing DNA damage at chromosomal levels (Fenech, 1993, Lando et al., 1998). MN is small chromatin bodies that appear in the cytoplasm by the condensation of acentric chromosome fragments or by whole chromosomes, lagging behind the cell division. Fenech, 1993 also briefly demonstrated that the MN are acentric chromosome fragment or whole chromosomes left behind during mitosis and appear in the cytoplasm of interphase cells as small additional nuclei. So, this is only biomarker, which allows the simultaneous evaluation of both clastogenic, and aneugenic effects in a wide range of cells.
Micronuclei Analysis Techniques In Vitro (Fenech, 1993) In Vivo (Due et al., 1984)
In Vitro:
MN analyzed by a simple process in which 0.5 ml venous blood drawn from volunteer after getting his or her consent for the purposed study and whole blood cultured in RPMI-1640 medium, supplemented with 2% phytohemagglutinin, 100IU/ml pencillin, 100mg/ml streptomycin, and 2mM L-glutamine. After incubation at 370 C for 44 hours, cytochalasin-B was added to culture at a final concentration of 6 mg/ml and finally culture incubated for 28 hours to collect binucleated cells. The cells then treated with 0.075M KCl for 5 minutes at room temperature and fixed in methanol and acetic acid (3:1) and stained with 5% Giemsa. Scoring and counting criteria was followed as per the procedure of Fenech, 2003.
In Vivo:
1-2 drops of capillary blood collected in a heparinized sedimentation tube by finger or earlobe (adult) or venous blood can also be used and add 0.3% methyl cellulose solution in a v/v ratio of 1:2 to 1:3 and mixed carefully, set the sedimentation tube in water bath at 370 C for about 30 to 60 minutes, centrifuge at 1000 rpm for 6 minutes. Fix the material on slide by the help of 100% methanol for 1 minute. Stain the slides in buffered Giemsa for 10 minutes. Allow to air dry. The MN will appear in small, spherical, separated chromatin masses in lymphocytes.
Scoring Criteria for micronuclei (MN): (Fenech, 2003)
Morphologically micronuclei are identical but smaller than the main nuclei. These shows the following characteristics:
- Round or oval in shape.
- Non-refractile so, easily distinguished from staining particles.
- Not linked or connected to the main nuclei.
- May touch but not overlap the main nuclei and micronuclear boundary should be distinguishable from the nuclear boundary.
- Same staining intensity of the main nuclei but occasionally staining may be more intense.
- Micronuclei are chromatin-containing structure in the cytoplasm surrounded by a membrane without any detectable link to the cell nucleus (Schiffmann and DeBoni, 1991).
References:
- Cortyman, R. I., Heddlem J. A., (1976). The production of micronuclei from chromosome aberration in irradicated cultures of human lymphocytes. Mutat. Res. 41: 321 – 332.
- Due, K. X., Ding, B. Y., Cai, Y. Y., Sun, Y, J., Zhou, P., Ma, G. J., and Wang, S., (1984). A study of micronucleus test by human skin puncture. Zool. Res. 5: 225 – 260.
- Fenech M (1993). The cytokinesis blocks micronucleus technique. A detailed description on the method and its application to genotoxicity studies in human Population. Mutat. Res. (285) 35-44.
- Fenech, M., (2003). The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxic studies in human populations. Mutat. Res. 285: 35 – 44.
- Fenech, M., and Morley, A. A., (1985a). Measurement of micronuclei in human lymphocytes. Mutat. Res. 148: 29 – 36.
- Fenech, M., and Morley, A. A., (1985b). The effect of donor age on spontaneous and induced micronuclei. Mutat. Res. 148: 99 – 105.
- Lando, C., Hagmar, L., Bonnassi, S., (1998). Biomarkers of cytogenetic damage in human and risk of cancer. The European Study Group on Cytogenetic Biomarkers and Ehalth. Med. Lav. 89: 124 – 131.
- Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA (1960). Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res. (20) 613-616.
- Mossman B T, Bignon J, Corn M, Seaton A, and Gee J B L. Asbestos: scientific developments and implications for public policy. Science. 1990; 247: 294 – 301.
- Okayasu, R., Takahashi, S., Yamada, S., Hi, T.K., and Ullrich, R. L., (1999). Asbestos and DNA double strand breaks, Cancer Res. 59: 298 – 300.
- Rahman, Q., Dopp, E., Lohani, M., and Sciffmann, D., (2000). Occupational and environmental factors enhancing the genotoxicity of asbestos. Inhala. Toxico: 12: 157 – 165.
- Ramanathan, A.L., and Subramanian, M., (2001). Present status of asbestos mining and related health problems in India – a Survey. Ind. Health. 39: 309 – 315.
- Schiffmann, D., and DeBoni, U., (1991). Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechnism by which micronuclei can arise. Mutat. Res. 246: 113 – 122.
Furquan Ahmad Ansari, Iqbal Ahmad, Mohd. Yunus and Qamar Rahman
Lucknow-226 001
E-mail: [email protected]
Mohd. Yunus – Dean, Department of Environmental Science,
Babasaheb Bhimrao Ambedkar University
(A Central University)
Lucknow-226 001 E-mail: [email protected] Mohd. Yunus – Dean, Department of Environmental Science, Babasaheb Bhimrao Ambedkar University (A Central University)
