Micronucleated erythrocytes as an assay to assess actions by physical and chemical genotoxic agents in Clarias gariepinus

doi:10.1016/0165-1161(94)90027-2

Mutation Research/Environmental Mutagenesis and Related Subjects

Volume 313, Issue 1, August 1994, Pages 1-5

https://doi.org/10.1016/0165-1161(94)90027-2 Get rights and content

Abstract

An in vivo study on the effects of both physical (γ-radiation) and chemical (mitomycin C) genotoxic agents was carried out with the catfish species, Clarias gariepinus. The fish were either exposed to γ-radiation at doses of 0–9 Gy or injected intraperitoneally with mitomycin C at concentrations of 0–2 mg/kg. Micronucleated erythrocytes were sampled from 0 to 60 days post treatment. Data obtained showed a time-dependent response of the induction of micronucleated erythrocytes with both genotoxic agents. A linear dose-dependent increase was observed 2–4 days after treatment. These data show the importance of sampling time in the micronucleus assay with Clarias gariepinus.

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Finally, in Nakano et al. (2003) study, mitomycin C elicited offspring malformations and germ cells mutations at concentrations ranging between 1 and 100 μM in mollusca. It has also induced dose and time-dependent micronuclei formation in fish with concentrations ranging between 0.25 and 10 mg/kg (Bahari et al., 1994; Das and Nanda, 1986; Winter et al., 2007). Tyrosine kinase inhibitors block the receptor tyrosine kinases that promote cell division and survival (Gustafson and Bailey, 2019).
Anticancer drugs in the aquatic environment have drawn a lot of attention in the last decade. Since wastewater treatment plants are inefficient at fully eliminating trace concentrations of anticancer drugs, these compounds are continuously discharged into the aquatic environment. Subsequently, non-target organisms such as the aquatic biota are directly exposed to a variety of anticancer drugs. To understand the potential impact on the aquatic organisms, a systematic review was conducted in compliance with the PRISMA guidelines. The results acquired from the 152 included studies were analysed and sorted into four categories: the impact of each included anticancer drug, the effect of metabolites, the effect of a mixture of drugs, and risk assessment. Findings showed that risk to the aquatic biota was unlikely to occur as the concentrations needed to induce effects were much higher than those detected in the environment. However, these data were based on acute toxicity and included only basic toxicity endpoints. The concentrations that produced significant effects were much lower when tested in the long-term or in multi-generational studies. Heterogeneity in results was also observed; this depended on the organism tested, the assessment adopted, and the endpoints selected. In this systematic review, an overall view of the research studies was generated by which all the variability factors to be considered were reported and recommendations to guide future studies were proposed.
Assessment of the cytotoxic and mutagenic potential of the Jialu River and adjacent groundwater using human-hamster hybrid cells
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We reasoned therefore that assessing the overall effects of these compounds will be facilitated by investigating the cytotoxicity and genotoxicity of the river water and its adjacent groundwater. Numerous in vivo tests on the genotoxicity of water samples were conducted, particularly using indigenous aquatic organisms such as fish (Bahari et al., 1994; Alsabti and Metcalfe, 1995; Minissi et al., 1996), sea urchins (Hose et al., 1983), mussels (Ericson et al., 2002; Klobucar et al., 2003), oysters (Burgeot et al., 1995), newts (Jaylet et al., 1986; Fernandez et al., 1993), and marine worms (Jha et al., 1995). Nevertheless, because of the insensitivity of aquatic organisms to genotoxic compounds and complexities involved in manipulating these organisms in the laboratory, the application of in vitro genotoxic tests of water samples is more helpful for investigating the presence and distribution of genotoxins (Ohe et al., 1993; Eckl, 1995; Schnurstein and Braunbeck, 2001).
The Jialu River in China has been seriously polluted by the direct discharge of industrial and domestic wastewater. The predominant contaminants of the Jialu River and its adjacent groundwater were recently investigated. However, the potential genotoxic impact of polluted water on human health remains to be clarified. Here, we used human–hamster hybrid (A_L) cells, which are sensitive for detecting environmental mutagens. We found that the cytotoxicity and mutagenicity of the groundwater in the Jialu River basin were influenced by the infiltration of the Jialu River. Hydrological periods significantly affected the cytotoxicity, but not the mutagenic potential, of surface and groundwater. Further, the mutagenic potential of groundwater samples located < 1 km from the Jialu River (S_M-2 water samples) was detected earlier than that of groundwater samples located approximately 20 km from the Jialu River (S_N water samples). Because of high cytotoxicity, the mutagenic potential of water samples from the Jialu River (S_M-1 water samples) was not significantly enhanced compared with that of untreated controls. To further assess the mutagenic dispersion potential, an artificial neural network model was adopted. The results showed that the highest mutagenic potential of groundwater was observed approximately 10 km from the Jialu River. Although further investigation of mutagenic spatial dispersion is required, our data are significant for advancing our understanding of the origin, dispersion, and biological effects of water samples from polluted areas.
Sensitivity of medaka (Oryzias latipes) to 4-nonylphenol subacute exposure; erythrocyte alterations and apoptosis
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Fish erythrocytes are favored because the frequency of alterations in both cytoplasm and nuclei can be a marker of cytotoxicity, genotoxicity, hemotoxicity, and oxidative stress (Bushra et al., 2002; Gomes et al., 2015; Joshi et al., 2002; Mekkawy et al., 2011; Sayed et al., 2017; Sayed et al., 2016c). The frequency of alterations in fish erythrocytes, the induction of MN, and their apoptosis have been studied after exposure UVA, pesticides, hydrocarbons, and toxins (Bahari et al., 1994; Bombail et al., 2001; Bushra et al., 2002; Mekkawy et al., 2011; Sayed et al., 2016c; Talapatra and Banerjee, 2007). Apoptosis is associated with signs of abnormal cell morphology including cell shrinkage, membrane blebbing and chromatin condensation (Cavas et al., 2005; Murakawa et al., 2001; Sayed and Hamed, 2017; Talapatra and Banerjee, 2007).
The present study was undertaken to assess the effects of the endocrine-disrupting compound; 4-nonylphenol (4-NP) in medaka (Oryzias latipes). The frequencies of erythrocyte alterations, apoptosis, and micronuclei were used as biological indicators of damage. Medaka were exposed 15 days to 4-NP at three sublethal concentrations (50, 80, and 100 μg/l 4-NP) and results compared with those of a previous study using catfish as an animal model. Exposure of medaka resulted in a dose-dependent increase in the frequency of erythrocyte alterations, apoptosis and micronucleus (MN). Many morphological alterations and nuclear abnormalities were observed, including acanthocytes, lobed nucleus, eccentric nucleus, fragmented nucleus, blebbed nucleus, binuclei, deformed nucleus, notched nucleus, hemolysed cells, crenated cells, teardrop-like cells, and schistocytes. Mortality was recorded after treatment with 80 and 100 μg/l 4-NP, indicating that medaka are more sensitive than catfish to 4-NP exposure. We concluded that, 4-NP causes several malformations in the shape and number of erythrocytes in medaka, indicating its genotoxicity.
Assessment of endosulfan induced genotoxicity and mutagenicity manifested by oxidative stress pathways in freshwater cyprinid fish crucian carp (Carassius carassius L.)
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Citation Excerpt :
Earlier, it has been emphasized that exposure of fish to genotoxic chemicals, for various interval of time, by the respiratory route following the absorption of chemicals through gill epithelium could be occurred (Rishi and Grewal, 1995; Farah et al., 2006). Our results of MN are in agreement with some earlier studies (Bahari et al., 1994; Ali et al., 2008). The frequency of CA observed in C. carassius after exposure to different concentrations of test chemical and standard genotoxic agent, cyclophosphamide, were significantly (p < 0.05) higher when compared to the negative control (Table 3), at all the exposure durations.
Over the past few decades, endosulfan, one of the polychlorinated pesticides still in use, has received considerable attention of a number of international regulations and restriction action plans worldwide. This study aimed to evaluate the cytogenetic effects of endosulfan using robust genotoxicity assays, along with the oxidative stress pathways in order to understand biochemical mechanism, in Carassius carassius L. The LC_50–96 h (95% confidence limits) value of endosulfan was 0.070 (0.046–0.093) ppm; and on its basis three test concentrations (sub-lethal I: 0.052, II: 0.035 and III: 0.017 ppm) were selected for 35 d in vivo exposure. The mean concentration of endosulfan in aquaria was always constant, when analyzed by dispersive liquid–liquid micro extraction (DLLME) followed by GC–MS. Autopsy was done on days 1, 2, 3, 4, 7, 14, 21, 28 and 35 of endosulfan exposure; the micronucleus formation (MN), authenticated by scanning electron microscopy, and chromosomal aberrations (CA), were induced significantly (p < 0.05) in all the treated groups, including positive control cyclophosphamide (4 ppm), when compared to negative control. Similarly lipid peroxidation (LPO) was induced significantly with the maximal at higher concentration (SL-I) on 4th day (722.45%; p < 0.01). Antioxidant biomarkers like glutathione reduced, superoxide dismutase and catalase also fluctuated significantly (p < 0.01) in all treatment groups. Collective findings demonstrated that genotoxic effects were invariably accompanied and correlated with increased oxidative stress and disturbance of antioxidant enzymes; and the MN and CA assays are useful tools in determining potential genotoxicity of aquatic xenobiotics and might be appropriate as a part of monitoring program.
Effects of 4-nonylphenol on blood cells of the African catfish Clarias gariepinus (Burchell, 1822)
2011, Tissue and Cell
Citation Excerpt :
The study of micronucleus test and abnormal erythrocytes morphology in fishes by various chemicals have been reported (Grisolia and Starling, 2001; Ferraro et al., 2004; Talapatra and Banerjee, 2007). Fish erythrocytes are especially favoured for micronucleus test (Bushra et al., 2002) and its feasibility has already been established in Clarias gariepinus (Bahari et al., 1994). It was stated by many authors that abnormal nuclear morphology is an indicator of genotoxic damage in fish (Bombail et al., 2001; Talapatra and Banerjee, 2007).
In the present work, the destructive effects of the 4-nonylphenol on one of the most economically important Nile fishes, namely African catfish (Clarias gariepinus) were studied. Apoptosis, erythrocytes alterations, micronucleus test and blood parameters count were used as biological indicators to detect those effects. After exposure to sublethal concentrations of 4-nonylphenol (0, 0.05, 0.08 and 0.1 mg/l), apoptotic red blood cells with many malformations and micronucleated erythrocytes were recorded. Decrease in the blood parameters such as red blood cells (RBCs), hemoglobin (Hb), package cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), platelets, white blood cells (WBCs), lymphocytes, basophils, monocytes and increase in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), neutrophils, eosinophils indicated the negative effects of 4-nonylphenol. It was concluded that, the 4-nonylphenol caused genotoxicity in erythrocytes with many malformations in shape and number indicated with other blood parameters.
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2010, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
Ultraviolet radiation (UVR) has many positive effects, but overexposure of organisms can generate alterations in DNA, either directly or indirectly, inducing mutagenic and cytotoxic lesions. The aim of this study was to evaluate the genotoxic effects of UVA–B/visible light in the broad-snouted caiman (Caiman latirostris), using the micronucleus (MN) test as a biomarker.
Seventy two juvenile caimans, approximately 5 months old, were maintained for during 3 months under total darkness, or under 8 or 16 h of daily exposure to artificial UV/visible light. MN test was applied before and after the experiment and the difference in MN frequencies was determined. Our results indicate significant increases in MN frequency with all treatments, compared to the basal (before experiment) values. Animals exposed to UV radiation showed a greater increase in MN frequency, compared to the animals exposed to total darkness (TD) treatment.
These results provide information about the possible harmful effects generated by sub-chronic exposure to UVR in zoos, reptile hobbyist and breeding programs, as well as the deleterious consequences of increased UV environmental impact on wild species such as the broad-snouted caiman.

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Mutation Research/Environmental Mutagenesis and Related Subjects

Abstract

References (17)

Micronucleus test in fish for monitoring the genotoxic effects of industrial waste products in the Baltic sea, Sweden

Comp. Biochem. Physiol.

High capacity in vitro micronucleus assay for assessment of chromosome damage: results with quinolone/naphthyridone antibacterials

Mutation Res.

Induction of micronucei in peripheral erythrocytes of fish Heteropneustes fossils by mitomycin C and paper mill effluent

Mutation Res.

Amphibian micronucleus test(s): a simple and reliable method for evaluating in vivo genotoxic effects of freshwater pollutants and radiations. Initial assessment

Mutation Res.

The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program

Mutation Res.

Micronuclei in lymphocytes with preserved cytoplasm: A method for assessment of cytogenetic damage in man

Mutation Res.

Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes

Mutation Res.

Micronuclei in red blood cells of the newt Pleurodeles waltl Michah: induction with X-rays and chemicals

Mutation Res.

Cited by (53)

Aquatic ecotoxicology of anticancer drugs: A systematic review

Assessment of the cytotoxic and mutagenic potential of the Jialu River and adjacent groundwater using human-hamster hybrid cells

Sensitivity of medaka (Oryzias latipes) to 4-nonylphenol subacute exposure; erythrocyte alterations and apoptosis

Assessment of endosulfan induced genotoxicity and mutagenicity manifested by oxidative stress pathways in freshwater cyprinid fish crucian carp (Carassius carassius L.)

Effects of 4-nonylphenol on blood cells of the African catfish Clarias gariepinus (Burchell, 1822)

Ultraviolet radiation-induced genotoxic effects in the broad-snouted caiman, Caiman latirostris