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Dna Damage And Cell Cycle Perturbation Analysis In The Fish Catla Catla (Ham.) Exposed To Gamma Radiation By Flow Cytometry

1618 words - 7 pages

Introduction

DNA damage is a proven biomarker of radiation exposure. Commonly used cytogenetic techniques for detecting DNA strand breakages include chromosome aberrations, micronucleus and comet assay under field and laboratory conditions. Flow cytometry (FCM), an instrumental tool has been widely used to quantify DNA damage. It offers the analysis of high number of nuclei with statistically reliable results in a short period of time. Ideally, all cells within an organism contain the same amount of DNA to maintain cellular homeostasis. DNA damage results from the breakage, chromosome rearrangement and interference with the normal segregation during cell division. Double strand breaks are ...view middle of the document...

1995; Dallas at al. 1998; Lingenfelser at al. 1997).
Alternately, appearance of aneuploid cell populations as a result of mitotic machinery defects are detectable by FCM analysis as additional peaks in the histogram, since these involve specific chromosomal deletions and rearrangements that are associated with several malignancies in chronic low dose exposures (Oshimura and Barrett, 1986). For instance, somatic cell aneuploidy has been found during the development of several types of cancers (Liang and Brinkley, 1995) considering as a useful prognostic marker in malignancies.
Cell cycle, a basic synchronizing event that takes place in three prominent phases to maintain the integrity of organism’s hereditary material and any provoking disturbances leads to the malfunctioning of several metabolic processes. This in turn reflects the organism’s survival in the ecosystem it inhabited. Using FCM, changes in cell cycle perturbations can be analyzed via categorizing the percentage of cell populations residing in each phase of the cell cycle and apoptotic cascade phenomenon. Since ionizing radiation is known to affect erythropoiesis and leucopoiesis in vertebrates (Watson et al. 1962; Finstad et al. 1969); advantage of nucleated erythrocytes in fish blood provide sufficient number of potentially affected cells for FCM analysis thereby considering them as a suitable model system.
Hence, in the present study gamma radiation delivered within a short duration (acute doses) and continuously over a relatively long period (protracted doses) induced DNA damage and cell cycle phase disturbances were studied in the peripheral erythrocytes of freshwater fish Catla catla using FCM.

2. Materials and methods
2.1. Experimental fish specimens

Freshwater fish Catla catla (Hamilton, Family: Cyprinidae) was chosen due to its sensitivity (Tilak et al., 1981) and high economic value in Asian market. Fingerlings weighing between 8-10 g and of length 8 ± 2.0 cm were procured from Government approved commercial fish farm and transported to the laboratory in oxygenated bags and released into 50L glass aquaria filled with dechlorinated tap water. They were then acclimatized for 30 days under laboratory conditions with natural photoperiod and fed with oil cake. The fecal matter and other waste materials were siphoned off daily to reduce ammonia content in water that was renewed once in two days with dechlorinated tap water. The water quality parameters were analyzed and maintained within the normal range (pH – 7.1±0.25, dissolved oxygen – 8.2±0.36 mg/l, Temp. –25 ± 1 °C and hardness– 220±0.0 ppm (CaCO3) respectively.

2.2. Exposure to radiation

A LD50/30 value of 22.375 Gy was deduced for this fish from previous study and a non-lethal dose of 5Gy was selected for the present investigation. The methodology of radiation exposure has been described in detail elsewhere (Anbumani and Mohankumar, 2012).

2.3. Blood cell collection and staining

Fish were anesthetized using...

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