Abstract
Understanding the effects of heavy metals in aquatic ecosystems is of significant importance due to their potential to bioaccumulate at various trophic levels and induce damage in DNA. Mercury is considered one of the most dangerous heavy metals, causing chromosomal breakage (clastogenic event) or spindle dysfunction (aneugenic event), that can lead to the formation of encapsulated chromatin into a separate smaller nucleus, generally referred to as a micronucleus. We evaluated the sensitivity of the micronucleus test in the neotropical cichlid Andinoacara rivulatus (Günther 1860). The fish were divided into four groups of 16 individuals, and each group was placed in separate aquaria (140 L) provided with filtered water and constant aeration. Fish were exposed to mercury chloride (HgCl2) at doses 0.1, 0.25, and 0.50 mg/Kg body weight, administered by intraperitoneal (IP) injection. Fish from the control group were injected with a physiologic solution. The following erythrocyte anomalies were identified: erythrocytes with micronuclei varying to some extent in size and position in the cytoplasm, blebbed nucleus, binucleated cell, nuclei showing a deep invagination toward the center (notched nuclei). Examination of blood smears demonstrated a higher level of micronucleus and notched erythrocytes in fish injected with HgCl2 than in the controls. There were significant differences in the frequency of micronucleated and notched erythrocyte among the groups exposed to mercury. Linear regression analysis revealed a positive relationship between the frequency of micronucleated and notched erythrocytes (P < 0.0001), with a moderately strong correlation coefficient (r=0.737). We propose that, in addition to the two so far known mechanisms of micronucleus formation (spindle apparatus damage and chromosomal ruptures), chromatin fragmentation in notched nuclei resulting from a combination of the cytotoxic effects of mercury and mechanical stress, may be a third mechanism of micronuclei genesis. It is emphasized that although the micronucleus test is a cheap and technically simple method that requires very basic equipment, direct counting of erythrocytes using a microscope is a time-consuming task because it requires counting a high number of cells (a minimum of 1,000 to 2,000 cells) per blood smear, leading to visual fatigue. We recommend, photographing the stained blood smears at several areas at random and scoring the digital images using Adobe Photoshop software as an easy way to evaluate a large number of erythrocytes for minimizing the visual fatigue caused by many hours of direct observation through a microscope
Highlights
Heavy metals released into aquatic ecosystems from industrial, agricultural, and domestic sources, municipal sewage treatment plants, and from other anthropogenic activities have the potential to bioaccumulate at various trophic levels and can induce damage in DNA (Mitchelmore & Chipman, 1998; Ohe, Watanabe, & Wakabayashi; 2004, Bolognesi, Perrone, Roggieri, Pampanin, & Sciutto, 2006; Isani et al, 2009; Sharifuzzaman et al 2016)
The analysis of peripheral blood smears of the studied fish showed that normal erythrocytes appeared with an oval shape and a centric ellipsoid nucleus with well-defined boundary (Fig. 1A)
The following nuclear abnormalities were found: small, non-refractive circular/ ovoid particles in the cytoplasm resembling a nucleus with respect to staining properties were considered an micronucleated erythrocytes (MNE) varying to some extent in size and position in the cytoplasm (Fig. 1B, Fig, 1C, Fig. 1D); blebbed nucleus appeared as a small evagination of the nuclear envelope resembling a micronucleus (Fig. 1E, Fig. 1F); binucleated cell contains two nuclei that are not attached and relatively similar in size (Fig. 1G); nuclei showing a deep invagination toward the center were considered an notched erythrocytes (NE) (Fig. 1 H)
Summary
Heavy metals released into aquatic ecosystems from industrial, agricultural, and domestic sources, municipal sewage treatment plants, and from other anthropogenic activities have the potential to bioaccumulate at various trophic levels and can induce damage in DNA (Mitchelmore & Chipman, 1998; Ohe, Watanabe, & Wakabayashi; 2004, Bolognesi, Perrone, Roggieri, Pampanin, & Sciutto, 2006; Isani et al, 2009; Sharifuzzaman et al 2016). Mercury is one of the most dangerous heavy metals, causing deleterious effects on biota (W.H.O., 1990; Buhl, 1997; Rocha et al 2011a; Anual, 2014; Nabi, 2014; Kandroo, Tripathi, & Sharma, 2015; Morcillo, Esteban, & Cuesta, 2017) This toxicant interferes with the formation of the mitotic spindle during cell division, causing the contraction of chromosomes, a delay in the division of the centromere, and a slower movement during anaphase, as well as chromosomal breakage (Skerfving, Hansson, & Lindsten, 1970; Catherine Ferens & United States, 1974; Thier, Bonacker, Stoiber, & Böhm, 2003). Research on clastogenic or mutagenic effects in neotropical fish has become increasingly common (Cestari et al, 2004; Rocha et al, 2011b; Zapata et al, 2016)
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