J. Indian Fish. Assoc., 32: 111-117, 2005 111 GENOTOXIC EFFECT OF NICKEL CHLORIDE AND ZINC SULPHATE ON FISH HYPOPHTHALMICHTHYS MOLITRIX S. Athikeshvan, S. Vincent and B. Velmurugan Unit of Environmental Health Biotechnology, PG and Research Department of Zoology, Loyola College, Chennai - 600 034, India. ABSTRACT The present investigation is to assess the genotoxic potential of nickel chloride and zinc sulphate on gill cells of silver carp Hypophthalmichthys molitrix. Fishes were exposed in sublethal concentration of nickel chloride 5. 7 mg/1 and zinc sulphate 6.8 mg/1, and sampled at 10, 20 and 30 days. Nickel chloride and zinc sulphate treated f"ishes exhibited an apparent increase in the aberration frequency and a decrease in the mitotic index as compared to control. Acentric fragment, chromatid break, endoreduplication, chromatid gap, centromeric fusion, ploidy, sticky plate, dicentric chromosome, clumping and partial sticky plates were some of the abnormalities observed. The chromosomal aberrations in the treated fishes were significant compared to con trot Keywords: Hypophthalmichthys molitrix:~ nickel chloride, zinc sulphate, gill cells, chromosomal aberration INTRODUCTION As fish may act as 'sentinel' organisms for indicating aquatic pollution, several species have been successfully used as test materials for detecting genotoxic activity in the aquatic environment (Kligerman et al., 1975; Hooftman, 1981; Manna and Mukherjee 1989). Fish, particularly those living in rivers or confined waters, also run the risk of direct or indirect exposure to various chemical mutagens present in the run-offs along with various toxicants, pesticides, industrial wastes, etc Guha and Khuda-Bukhsh (2003). Analysis of metaphase chromosomes in fish for the occurence of chromosome aberrations and sister-chromatid (SCE) in order to detect as well as quantify the extent of genotoxicity or point mutation induced by an agent has proven to be useful in fish models (Kligerman 1982, Krishnaja and Rege 1982, Manna, 1984). Manna et al., (1985); Monoharan and Prabhakaran (1994) were reported the genotoxic nature of heavy metal pollutants in fish. The genotoxic effects of cadmium chloride (CdCh) and Azadirachtin (Aza) were assessed singly and conjointly in a
112 GENOTOXIC EFFECT OF NICKEL CHLORIDE AND ZINC SULPHATE fish, Oreochromis mossambicus, with endpoints such as chromosome aberrations (Chandra and Khuda B ukhsh, 2004 ). The present investigation study was designed to find the genotoxicity induced by nickel chloride and zinc sulphate in the fish H. molitrix. MATERIAL AND METHODS Healthy specimens of H. molitrix measuring 5.5 ± 0.5 em length and 6.0 ± 1.0 g weight were collected from Poondy reservoir near Chennai and brought to the laboratory in well-aerated containers. The fish were acclimatized to the laboratory conditions for 15 to 20 days prior to experimentation. Acute toxicity assay were carried and 96 h LCso value found to be 57 mg/1 for nickel chloride and 68 mgll for zinc sulphate. For genotoxicity assay fishes were exposed to sublethal concentration of nickel chlor-ide 5.7 mg/1 and zinc sulphate 6.8 mg/l individually. Fishes are sampled after 10, 20 and 30 days of exposure. The fishes were sacrificed and the gill was dissected out and the slides were screened for metaphase plates under the light microscope and microphotographs were taken using Carl Zeiss Photomicroscope. RESULTS AND DISCUSSION H. molitrix has 48 (2n=48) chromosomes. The diploid chromosome number is in conformity with earlier report (Khuda and Chakrabarti 1996; Yao et al. 1994). The varied frequency of chromosomal aberrations due to exposure of H. molitrix to nickel chloride were found to be indistinct chromosome morphology, sticky plate, chromatid gap, nucleolar organizer region, centromic fusion, dicentric chromosome clumping and ring chromosome (Fig.1 ). Zinc sulphate treated chromosome were found to be ring chromosome condensed chromosome short arm, polyploidy, dicentric chromosome, sticky plate, nucleolar organizer region, endoreduplication and centromic fusion (Fig.2). The frequencies of chromosomal aberrations were found to be decrease with in increase in duration in both nickel chloride and zinc sulphate treated fishes (Table 1). According to Cavas and Ergene-Gozukara (2003), differences in the micronucleus frequencies with time seem to be related to cell kinetics and replacement. One other reason could be that the gill cell are directly and continuously exposed to contaminated water. Nickel chloride treated fishes exhibited higher chromosomal aberrations compared to zinc sulphate treated fishes. Chromosomal aberrations were analysed using t-test, and values are significant at p 0.05 level compared to the control. the present finding closely agrees with the findings of Manna et al. (1985) and Manoharan andprabhakaran (1994) A1 Sabti, (1985a,b ). Most of the toxic chemicals that produce genotoxic effects have been known to form reactive oxygen species as well as
~ TABLE 1: Frequency of chromosomal aberrations induced by nickel and zinc in the gill cells of Hypophthalmichthys molitrix. Treatment Metaphase Total No. of AF CB ED CG CF PL SP DC PS t-value (days) plate studied Aberrations C--o 10 100 11 13 4 5 3 4 4 2 1 47 Nickel 20 100 9 8 3 4 2 4 5 2-37 30 100 7 6 2 3 1 3 4 1 2 29 10 100 10 11 4 5 3 4 3 2 1 43 Zinc 20 100 7 8 3 5 2 4 5 1 1 36 1.98 1.96 1.92 1.97 1.94 en ~ I :A: m en I ~ z rn _, )::. r- 30 100 6 6 2 1 2 2 4 1 1 25 1.90 Control 100 2 2-1 - 1 - - - 6. Af- Acentric Fragment; CB - Chromatid Break; ED - Endo Reduplication; CG - Chromatid gap; CF - Centromeric Fusion; PL- Ploidy, SP- Sticky Plate, DC- Dicentric Chromosome; PS - Partial Sticky Plate. *Values are signzfigant at p 0.05% confidence limit. -1. -' (..)
114 GENOTOXIC EFFECT OF NICKEL CHLORIDE AND ZINC SULPHATE Fig. 1: Chromosomal aberrations induced by nickel in Hypophthalmichthys molitrix A) Control chromosome B) Indistinct chromosome morphology C) Sticky plate D) Chromatid Gap E) Nucleolar Organizer Region F) Centromic fusion G) Dicentric Chromosome H) Clumping I) Ring chromosome
S. ATHIKESHVAN ET AL., 115 F Fig. 2: Chromosomal aberrations induced by zinc in Hypophthalmichthys molitrix A) Control chromosome B) Ring chromosome C) Condensed chromosome with short arm D) Polyploidy E) Dicentric Chromosome F) Sticky plate G) Nucleolar Organizer Region H) Endoredupiication I) Centromic fusion electrophilic free-radical metabolites that interact with DNA to cause disruptive changes (Chandra and Khuda-Bukhsh, 2004). Ni can bind to DNA and proteins in cells in vitro and to chromatin in vivo. Such binding to macromolecules could be correlated to the ability of Ni compounds to interfer with DNA synthesis and to induce slight increases in chromosome alterations, as well as its mutagenic action (Morita et al., 1991; Sarkar, 1995). It has also been suggested that Ni-induced abnormal DNA repair may be a mechanism for carcinogenesis (Au et al., 1994; Hartmann and Hartwig, 1998). In
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