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MN & CA: Biomarkers for Genotoxicity

Furquan Ahmad Ansari, Iqbal Ahmad, Mohd. Yunus and Qamar Rahman

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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.

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