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Biological Degradation and Detoxification of Toxic Contaminants in Leachate
Published in M.H. Fulekar, Bhawana Pathak, Bioremediation Technology, 2020
Indu Shekhar Thakur, Juhi Gupta, Pooja Ghosh
Genotoxicity is measured to analyze any breakage in DNA strands, excision and repair of DNA strands, or any formation of alkali labile salts which may occur due to exposure to various contaminants. The comet assay has been frequently employed to evaluate the genotoxicity of environmental samples. Unlike other genotoxic assays, the comet assay is more advantageous and has a high sensitivity to even detect low levels of DNA damage, less cell requirement per sample, short time requirement, and low cost. Additionally, this assay is quite flexible to detect different DNA damages and modifies enough to adapt to different experimental settings (Tice et al., 2000). It is found that genotoxic effects are occurring due to the generation of free radicals and oxidative damage reported in the affected organs like the heart, spleen, liver, and brain (Li et al., 2006a, b). Such tests have been applied to observe the toxicity of landfill leachate in erythrocytes from the gill cells of goldfish (Carassius auratus) (Deguchi et al., 2007). Thus, the comet assay is a promising test to evaluate the gene-based effects of contaminants on DNA and other related organs.
In Vitro: Limitations of the Main Cytotoxicity Assays
Published in Vineet Kumar, Nandita Dasgupta, Shivendu Ranjan, Nanotoxicology, 2018
Montserrat Mitjans, Daniele Rubert Nogueira-Librelotto, María Pilar Vinardell
To study the interference of nanoparticles with 2′,7′-dichlorofluorescein fluorescence measurements, different dilutions of the fluorescent compound were prepared with nanoparticle dispersions and their fluorescence emission was determined. All the nanoparticle types tested interfered with 2′,7′-dichlorofluorescein, so to minimize this, the classical assay protocol was modified and the nanoparticle dispersions were removed by washing the cell monolayers prior to incubation with 2′,7′-dichlorodihydrogluorescein diacetate (Kroll 2012). One important aspect to study is the potential of nanoparticles to damage DNA. One of the mechanisms proposed to explain the capacity of nanoparticles to cause DNA damage is their capacity to cause oxidative stress. The comet assay is a sensitive method of detecting DNA strand breaks as well as oxidatively damaged DNA at the level of single cells, and it is commonly used in nanogenotoxicology. However, there are concerns regarding possible interactions between nanoparticles and the comet assay which a recent review critically discussed. The authors concluded the assay is reliable, considering that if an interaction occurs between nanoparticles and DNA, it is a very localized effect that does not affect the results. Under normal experimental conditions, additional damage to DNA is not observed to any significant extent. In the case of photocatalytic activity such as that of anatase titanium dioxide, the experiment should be performed avoiding light from fluorescent tubes to ensure there is no activation of the titanium dioxide (Karlsson 2015).
Cationic cellulose nanocrystals as sustainable green material for multi biological applications via ξ potential
Published in Journal of Biomaterials Science, Polymer Edition, 2023
Sara Mohamed Mahmoud, Safwat Hassan Ali, Mohamed M. A. Omar
Genotoxic damage of DNA was assessed by the comet or alkaline single cell gel electrophoresis (SCGE) technique with certain modifications [22]. The comet assay is a simple and sensitive method for detecting DNA damage in individual eukaryotic cells. Using agarose gel electrophoresis, quantify DNA fragmentation [23]. The DNA damage is quantified by measuring the displacement between the genetic material of the nucleus ('comet head’) and the resulting 'tail’. Tail Moment and Tail DNA% are the two most commonly parameters used to analyze Comet assay results. At least 50–100 cells should be analyzed per sample. The Tail Moment has been suggested to be an appropriate index of induced DNA damage in considering both the migration of the genetic material as well as the relative amount of DNA in the tail. The comet images were analyzed with the comet score analysis system. For each cell, the length of DNA migration (tail length) was measured in pixel (PX) from the center of the nucleus to the end of the tail. The percentage of DNA in the tail was estimated by measuring the total intensity (fluorescence) in the cells, which was taken as 100%, and determining what percentage of this total intensity corresponded to the intensity measured only in the tail. The tail moment, expressed in arbitrary units, was calculated as: tail length X percentage of migrated DNA/100, cells observation at 40× magnification using an fluorescence microscope (ZEISS) connected through a black and white camera to an image analysis system (Comet Assay II; Perceptive Instruments Ltd, UK).
Environmental transformation and nano-toxicity of engineered nano-particles (ENPs) in aquatic and terrestrial organisms
Published in Critical Reviews in Environmental Science and Technology, 2020
Qumber Abbas, Balal Yousaf, Habib Ullah, Muhammad Ubaid Ali, Yong Sik Ok, Jörg Rinklebe
Comet assay is a widely used technique to assess the damage in DNA structure. Even low exposure concentration of TiO2 ENPs (12.5 mg L−1) reduced the mitotic index and induced the chromosomal aberration in the onion roots (Allium cepa L.). Microscopic techniques along with comet assay confirmed the chromosome breakage, micronuclei formation, sticky and multipolar chromosomes (Pakrashi, Jain, et al., 2014). In addition of comet assay, DNA laddering assay can be used to investigate the DNA internucleosomal cleavage and plants apoptotic cell death upon interaction with CNTs (Ghosh, Bhadra, Adegoke, Bandyopadhyay, & Mukherjee, 2015). These studies indicate that ENPs upon interaction with plants induced cyto-genotoxic effects and regulate the expression levels of different genes involved in physiological and biochemical processes (Table 2). However, further in-depth studies are required in order to understand the severity of DNA damage, mechanism of plants recovery from DNA damage, and intergenerational transfer of traits to next progeny under the influence of nano-genotoxicity. Moreover, proteomic, and metabolomic studies should also be conducted to understand the complex ENPs interaction with different plant species.
Could fluoride be considered a genotoxic chemical agent in vivo? A systematic review with meta-analysis
Published in International Journal of Environmental Health Research, 2023
Giovana Wagner Branda Drummond, Wilton Mitsunari Takeshita, Glaucia Monteiro de Castro, Jean Nunes dos Santos, Patricia Ramos Cury, Ana Claudia Muniz Renno, Daniel Araki Ribeiro
Micronucleus (MN) is an assay that identifies a chromosome or a fragment of chromosome that during mitosis was not included in the nucleus of the cell. Its size corresponds to 1/3 to 1/5 of nucleus size and is located near it. The appearance of MN is a natural process of the organism that occurs with aging itself, but exposure to xenobiotics and some diseases can increase the frequency resulting in DNA damage (Sommer et al. 2020). Comet assay (SCEG) is a method that quantifies DNA breakage. It consists in the movement of DNA fragments when subjected to electrophoresis. The lower molecular weight fragments are dragged in the anionic direction, resulting in a comet shape. The length of the tail determines the damage to the genetic material (Lu et al. 2017; Møller et al. 2020). There are two types of comet assay. The most specific is the alkaline one for detecting damage in single strands, double strands, incomplete repair regions and alkali-labile. Neutral comet assay only detects double-stranded DNA damage (Singh 2016). The sister chromatid exchange (SCE) test is able to determine the impact of clastogens effects on whole chromosomes. During the S phase of the cell cycle, occurs crossing over of genetic material between two chromatids in the same chromosome. Exposure to a genotoxic compound can lead to an increase in SCE. This technique evaluates DNA damage quantitatively and qualitatively (Wilson Iii and Thompson 2007; Kwasniewska and Bara 2020). Chromosomal aberration (ABS) test is used to identify toxic substances with the potential to induce abnormalities to chromosomes, such as chromosomal breaks or translocation of sister chromatids and thereby result in damage to genetic material, mutations, and cancer (Turkez et al. 2017).