Micronuclei Assay: A Suitable Tool for Evaluating the Heavy Metals Induced Genotoxicity in Fish, Labeo rohita

Labeo rohita was selected for this experiment. Fish were procured from Fish Seed Hatchery, Faisalabad and live transferred to wet laboratory at Fisheries Research Farm, University of Agriculture Faisalabad. Fish were initially acclimatized to laboratory environment for a period of 14 days and then fish (n=10) of 120-day age (average weight, 16.21±0.35; length,115.41±2.26) were shifted to 100-L glass aquarium. The LC50 of chromium(Cr)+cobalt(Co) mixture for 96-hr for L. rohita was calculated as 39.92 mgL-1 (Batool and Javed, 2015). Fish were treated with different sub-lethal doses viz. 2/3rd, 1/ 3rd,1/4th and 1/5th of LC50 of Cr+Co mixture calculated as 26.61, 13.30, 19.98 and 7.98 mgL-1, respectively. Water was partially changed throughout the study period. Fish were treated for one month and blood samples were collected after seven days interval. Fish (n=10) were maintained in clean water considered as negative control (NC) (Kousar and Javed, 2015). The cyclophasphamid was used as a positive control (PC). Fish were fed with diet available commercially at 2% body weight. The water quality parameters such as water hardness (225 mgL-1), temperature (28ᵒC), pH (7.0) and dissolve oxygen (5ppm) were monitored for whole study.

Blood from the fish caudal vein was collected and instantly smeared a drop on slide. The smears were immediately fixed in methanol for 10 minute and left over for air dry, and finally stained with wright-giemsa stain for 8 minutes (Barsiene et al., 2004). Scoring of micronuclei and deshape nuclei were performed (per 1,000 cell) on coded slides using a binocular microscope according to the criteria described by Fenech et al. (2003). Following formulae was used to calculate the MN frequency.

The experiment was performed with three independent replicates for each treatment. Data were expressed as mean (±SE) and analyzed by non-parametric Mann-Whitney U-test (Steel et al., 1996).

Results showed that test dose 26.61 mgL-1 induced significantly higher mean MN followed by 13.30 mgL-1, 19.98 mgL-1, 7.98 mgL-1, PC and NC (Fig. 1C). However, the result of DN showed minor difference. The frequency of DN in RBCs was maximum in fish exposed to 26.61 mgL-1 dose followed by the order: 13.30 mgL-1> 19.98 mgL-1> PC > 7.98 mgL-1> NC (Fig. 1B). Micronuclei and deshaped nuclei frequency differ with the exposure period as 28>21>14>7 days (Table I).

Aquatic environment has been contaminated due to presence of various toxicants released from industries and agricultural activities (Isani et al., 2009). Aquatic pollutants include heavy metals which may enter in aquatic ecosystem through natural and anthropogenic sources. Sub-lethal exposure of these metals to aquatic organisms results in amassment of metals in tissues that cause unfavorable effects not only in the exposed animal, but also in humans through food chain (IARC, 1993). Various toxicants in contaminated water can induce damage to genetic material of aquatic life and produce geno-toxic effects. Aquatic animals especially, fish is a good indicator to study the heavy metals produced genotoxic and mutagenic damage such as DNA strand breakages (Pruski and Dixon, 2002).

In recent investigation, the ability of Cr+Co mixture to produce micronuclei (MN) and deshape nuclei in erythrocytes of L. rohita varied due to test doses and exposure period. The formation of MN and deshaped nuclei increased during first 21-day, after that it was decreased. These nuclear abnormalities can be rapidly defeated by DNA repair mechanisms (Mateuca et al., 2006). Mixture of Cd+Zn significantly increased the formation of MN and nuclear abnormalities (NA) in the erythrocyte of O. niloticus in a duration dependent manner (Abu-Bakar et al., 2014). Similarly, Jiraungkoorskul et al. (2007) also observed the time-dependent effects in RBCs of fish exposed to cadmium, copper, lead, and cadmium chloride. Potential of cadmium and mercury to induce MN and NA in Phoxinus phoxinus was recorded by Ayllon and Gracia-Vazquez (2000).

Several authors reported the Cd induced MN in RBCs of fish like C. paleatus, C. carpio and C. gibelio (Cavas et al., 2005) and A. anguilla (Sanchez-Galan et al., 2001). According to Udroiu (2006) the production of micronucleus in blood varies due to species and exposure period. A significantly positive correlation between metal

Small alphabet superscripts show the difference between treatments within the same row While capital alphabet shows significant (P < 0.05) among different durations of exposure within the same column.

concentration and frequency of NA in O. niloticus was noted by Summak et al. (2010). A dose (arsenic) reliant raise in MN and NA frequency in Channa punctatus and Carassius auratus was observed by Kumar et al. (2013). Kousar and Javed (2016) also reported the arsenic induced micronuclei and de-shaped nuclei in fish species viz. L. rohita, C. idella, C. catla and C. mrigala. Arsenic, mercury and copper also can induced genotoxicity in Channa punctata (Yadav and Trivedi, 2009). Kousar et al. (2018) observed the concentration dependent higher micronuclei frequency in RBCs of Cirrhina mrigala. Chromium induced genotoxicity in term of micronuclei in L. rohita was observed by Parveen et al. (2011). The abattoir effluent also induced the frequency of MN in a time reliant manner was observed by Alimba et al. (2015). Rasal et al. (2011) also reported the chromium induced genotoxicity in Labeo rohita.

The present study indicated that Cr+Co mixture is a genotoxic agent for Labeo rohita under different concentrations. Micronucleus test is a sensitive and rapid method to detect the effect of heavy metal pollution in aquatic environment.

The authors declare there is no conflict of interest.