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Genotoxicity of Functionalized Nanoparticles
Published in Vineet Kumar, Praveen Guleria, Nandita Dasgupta, Shivendu Ranjan, Functionalized Nanomaterials II, 2021
Varsha Dogra, Gurpreet Kaur, Rajeev Kumar, Sandeep Kumar
This is the process of engulfment of a solid particle, typically by macrophages, neutrophils, dendritic cells, monocytes, and mast cells which is instigated by particle opsonization and leads to the stimulation of F-actin-driven pseudopods that ingest the solid particle and forms phagosome. This process can be inhibited by the action of cytochalasin D (Geiser et al. 2005) which blocks actin polymerization (Bronson 1998) that stops the action of pseudopodia. Neutrophils, macrophages, and monocytes are specialized phagocytes, other cells like epithelial cells, fibroblast, etc. can also uptake the solid particles by the process called pinocytosis (Mellman 1996). This process takes places at the site of clathrin-coated pits that are found in most animal cells and includes the intake of particles having a size less than 200nm, along with fluids surrounding the particles.
Polyethylene Glycol Polyester Block Copolymers: Biocompatible Carriers for Nanoparticulate Drug Delivery
Published in Dan Peer, Handbook of Harnessing Biomaterials in Nanomedicine, 2021
David S. Hart, Yunqi Zhao, M. Laird Forrest
As this macrophage uptake would represent one mode of the micelles clearance in vivo, it is also important to understand how these micelles interact with other cells. This question has typically been addressed through the use of fluorescent dyes or radioactive compounds. Both of these approaches were used to demonstrate that the cellular uptake of PEO44-PCL20 micelles in a PC 12 cell line was temperature and pH dependent [76] in a manner indicative of endocytosis uptake mechanisms. Mahmud et al. studied the uptake of various PEO-PCL micelles with different PCL block lengths in MCF-7 breast cancer cells [21]. These micelles were loaded with a lipophilic fluorescent dye, DiI, and the mechanism of endocytic uptake was investigated with chlorpromazine and cytochalasin B. An optimal PCL block length of 13 kDa, and PEO block length of 5 kDa demonstrated the highest cellular uptake. Chlorpromazine is known to inhibit clathrin-mediated endocytosis by reducing clathrin-coated pit formation at the cellular membrane, while cytochalasin B inhibits phagocytosis through disruption of microfilament bundles at the cell membrane. Both endocytosis inhibiting agents reduced uptake at the optimal block lengths of PEO and PCL. Incubation of MCF-7 cells at 4°C in the absence of these agents also reduced uptake. This suggests an energy-dependent uptake mechanism, which further supports both clathrin-mediated endocytosis and phagocytosis.
Pollution
Published in Brian D. Fath, Sven E. Jørgensen, Megan Cole, Managing Global Resources and Universal Processes, 2020
Vera Lucia S.S. de Castro, Paola Poli
After exposure to a test substance and addition of cytochalasin B for blocking cytokinesis, cell cultures are grown for a period sufficient to allow chromosomal damage to lead to the formation of micronuclei in bi- or multinucleated interphase cells. Harvested and stained interphase cells are then analyzed microscopically for the presence of micronuclei. Micronuclei are scored in those cells that complete nuclear division following exposure to the test item. Additionally, the cells are classified as mononucleates, binucleates, or multinucleates to estimate the proliferation index as a measure of toxicity.
Cytogenotoxic evaluations of leaves and stems extracts of Rubus rosifolius in primary metabolically noncompetent cells
Published in Journal of Toxicology and Environmental Health, Part A, 2023
Ana Paula Oliveira de Quadros, Isabel Bragança Baraldi, Marcel Petreanu, Rivaldo Niero, Mario Sergio Mantovani, Isabel O’Neill De Mascarenhas Gaivão, Edson Luis Maistro
This test was performed based upon the protocol described by Fenech (2000) and revised and standardized by OECD (2016). The cell culture was carried out in 25 cm2 flasks. To carry out the test, whole blood (0.5 ml) was used and 4.5 ml culture medium (RPMI) supplemented with 15% FBS and 170 µl phytohemagglutinin (PHA −3.4%) were added to stimulate lymphocyte mitogenesis. Subsequently, the cell culture was kept in a CO2 incubator at 36.5°C and homogenized at T = 24 hr. Forty-four hours following initiation of cultures, cells were treated with extracts at concentrations of 0.01, 0.1, 1, 10, 20, or 100 μg/ml, and also with the positive control (150 µM MMS) and the negative control (1% Tween 80). Treatments at this time ensure that cells in G1, S, G2, and M are exposed to the test substance. Four hours after treatments, 6 μg/ml cytochalasin B was added to each culture flask to block cytokinesis. Strictly 24 hr after the addition of cytochalasin B (T = 72 hr), the cells were collected and fixed. Harvesting was performed by centrifugation (5 min at 850 g), and the pellet was resuspended in ice-cold hypotonic KCl solution (0.075 M for 5 min). Subsequently, after centrifuging and discarding the supernatant, the cells were fixed with 5 ml of ice-cold methanol:acetic acid (3:4) (v/v) plus 3 drops of formaldehyde (37%) to preserve the cell cytoplasm (repeated fixation twice after centrifuging, and a last fixation with methanol:acetic acid (5:1)).
The effect of arsenic, cadmium, mercury, and lead on the genotoxic activity of Boletaceae family mushrooms present in Serbia
Published in Journal of Toxicology and Environmental Health, Part A, 2023
Marija Dimitrijević, M. Stanković, J. Nikolić, V. Mitić, V. Stankov Jovanović, G. Stojanović, D. Miladinović
All cultures were incubated at 37°C for 19 hr and subsequently all cultures were rinsed with pure medium and transferred to 5 ml fresh RPMI 1640 medium (RPMI 1640 Medium + GlutaMAX + 25 mm HEPES) and incubated for an additional 72 hr. After incubation, cytochalasin B was added to the samples, at concentration of 6 µg/ml, and incubation continued for another 24 hr. After incubation, cells were washed with 0.9% NaCl, collected by centrifugation and treated with hypotonic solution (0.56% KCl + 0.9% NaCl, mixed in equal volumes) at 37°C. The cell suspension is prefixed in methanol /acetic acid 3:1, (v/v) washed three times with fixative and placed onto a clean slide. The slides were air dried and stained with alkaline Giemsa. At least 1000 binuclear cells per sample were analyzed, according to the criteria of Countryman and Heddle (1976) and Fenech (1993b).
Genotoxic risk in occupational exposure to petrol and its amelioration by vitamin C and vitamin E
Published in Archives of Environmental & Occupational Health, 2022
Amrin Shaikh, Puranjay Chandel, Divya Chandel
Whole blood cultures were setup according to the method of Fenech9 with slight modifications to perform Micronucleus assay, where 7 mL of RPMI 1640 media (pre-supplemented with 10% fetal calf serum) was taken and 0.1 mL of phytohemagglutinin was added followed by 0.5 mL of heparinized whole blood. At 44 h, 10 µL of cytochalasin-B was added and the cultures were harvested at 72 h. Culture tubes were centrifuged at 2000 rpm for 15 min and the supernatant was discarded. The cells in the pellet were suspended by adding 6 mL of hypotonic solution (0.56% KCl) and incubated in a water bath at 37 °C for 20 min. Cells were fixed in chilled fixative (1:3, acetic acid:methanol) and culture tubes were kept in an ice bath for 20–30 min. After two changes of fixative, cells were suspended in a small volume of fixative and slides were prepared by gently dropping this cell suspension onto pre-cleaned, chilled, wet slides, dried on a slide warmer, and stained with 2% Giemsa Stain.