Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Ultrastructural Changes in Autopsy Tissues of COVID-19 Patients.

INTRODUCTION: The COVID-19 pandemic resulted in substantial morbidity and mortality across the world. The prognosis was found to be poor in patients with co-morbidities such as diabetes, hypertension, interstitial lung disease, etc. Although biochemical studies were done in patient samples, no study has been reported from the Indian subcontinent about ultrastructural changes in the vital organs of COVID-19 patients. The present study was, therefore, conducted to understand the ultrastructural changes in the lung, liver, and brain of the deceased patients. METHODS: The present study was conducted on samples obtained from reverse transcription-polymerase chain reaction (RT-PCR)-positive patients who were admitted to a tertiary care hospital in Western India. Core needle biopsies were done in eight fatal cases of COVID-19. The samples were taken from the lungs, liver, and brain and subjected to light microscopy, immunohistochemistry (IHC), and transmission electron microscopy (TEM). Clinical details and biochemical findings were also collected.  Results: The study participants included seven males and one female. The presenting complaints included fever, breathlessness, and cough. Light microscopy revealed diffuse alveolar damage in the lungs. Further, a positive expression of SARS-CoV-2 nucleocapsid protein was observed in the pulmonary parenchyma of five patients. Also, the TEM microphotograph showed viral particles of size up to 80nm localized in alveolar epithelial cells. However, no viral particles were found in liver or brain samples. In the liver, macrovesicular steatosis and centrizonal congestion with loss of hepatocytes were observed in light microscopy. CONCLUSION:  This is the first study in the Indian population showing the in-situ presence of viral particles in core biopsies from fatal cases of COVID-19. As evident from the results, histology and ultrastructural changes in the lung correlated with the presence of viral particles. The study revealed a positive correlation between the damage in the lungs and the presence of viral particles.

The Comet Assay: Assessment of In Vitro and In Vivo DNA Damage.

Anthropogenic activities, indiscriminate and rapid industrialization as well as pursuance of a better life has led to an increase in the concentration of chemicals, like pesticides, automobile exhausts, and new chemical entities, in the environment, which have an adverse effect on all living organisms including humans. Sensitive and robust test systems are thus required for accurate hazard identification and risk assessment. The Comet assay has been used widely as a simple, rapid, and sensitive tool for assessment of DNA damage in single cell from both in vitro and in vivo sources as well as in humans. The advantages of the in vivo Comet assay are its ability to detect DNA damage in any tissues, despite having non-proliferating cells, and its sensitivity to detect genotoxicity. The recommendations from the international workshops held for the Comet assay have resulted in establishment of guidelines, and the OECD has adopted a guideline for the in vivo Comet assay as a test for assessing DNA damage in animals. The in vitro Comet assay conducted in cultured cells can be used for screening large number of compounds and at very low concentrations. The in vitro assay has also been automated to provide a high throughput screening method for new chemical entities, as well as in environmental samples. This chapter details the in vitro Comet assay using the 96-well plate and in vivo Comet assay in multiple organs of the mouse.

The Comet Assay in Human Biomonitoring.

Human biomonitoring studies aim to identify potential exposures to environmental, occupational, or lifestyle toxicants in human populations and are commonly used by public health decision makers to predict disease risk. The Comet assay measures changes in genomic stability and is one of the most reliable biomarkers to indicate early biological effects and therefore accepted by various governmental regulatory agencies. The appeal of the Comet assay lies in its relative simplicity, rapidity, sensitivity, and economic efficiency. Furthermore, the assay is known for its broad versatility, as it can be applied to virtually any human cell and easily adapted in order to detect particular biomarkers of interest, such as DNA repair capacity or single and double-strand breaks. In a standard experiment, isolated single cells are first embedded in agarose, and then lysed in high-salt solutions in order to remove all cellular contents except the DNA attached to a nuclear scaffold. Subsequent electrophoresis results in accumulation of undamaged DNA sequences at the proximity of the nuclear scaffold, while damaged sequences migrate toward the anode. When visualized with fluorochromes, these migrated DNA fragments resemble a Comet tail and can be quantified for their intensity and shape according to internationally drafted guidelines.

Multicolor Laser Scanning Confocal Immunofluorescence Microscopy of DNA Damage Response Biomarkers.

DNA damage through endogenous and environmental toxicants is a constant threat to both a human's ability to pass on intact genetic information to its offspring as well as in somatic cells for its own survival. To counter these threats posed by DNA damage, cells have evolved a series of highly choreographed mechanisms-collectively defined as the DNA-damage response (DDR)-to sense DNA lesions, signal their presence, and mediate their repair. Thus, regular DDR signaling cascades are vital to prevent the initiation and progression of many human diseases including cancer. Consequently, quantitative assessment of DNA damage and response became an important biomarker for assessment of human health and disease risk in biomonitoring studies. However, most quantitative DNA damage biomarker techniques require dissolution of the nuclear architecture and hence loss of spatial information. Laser scanning confocal immunofluorescence microscopy (LSCIM) of three-dimensionally preserved nuclei can be, quantitative and maintain the spatial information. Here we describe the experimental protocols to quantify individual key events of the DDR cascade in three-dimensionally preserved nuclei by LSCIM with high resolution, using the simultaneous detection of Rad50 as well as phosphorylated H2AX and ATM and in somatic and germ cells as an example.

In Silico Approaches in Predictive Genetic Toxicology.

Genetic toxicology testing is a weight-of-evidence approach to identify and characterize chemical substances that can cause genetic modifications in somatic and/or germ cells. Prediction of genetic toxicology using computational tools is gaining more attention and preferred by regulatory authorities as an alternate safety assessment for in vivo or in vitro approaches. Due to the cost and time associated with experimental genetic toxicity tests, it is essential to develop more robust in silico methods to predict chemical genetic toxicity. A number of in silico genotoxicity predictive tools/models are developed based on the experimental data gathered over the years. These in silico tools are divided into statistical quantitative structure-activity relationships (QSAR)-based approaches and expert-based systems. This chapter covers the state of the art in silico toxicology approaches and standardized protocols, essential for conducting genetic toxicity predictions of chemicals. This chapter also highlights various parameters for the validation of the prediction results obtained from QSAR models.

Correction: Topical Application of Ochratoxin A Causes DNA Damage and Tumor Initiation in Mouse Skin.

[This corrects the article DOI: 10.1371/journal.pone.0047280.].

TiO2 NPs Induce DNA Damage in Lymphocytes from Healthy Individuals and Patients with Respiratory Diseases-An Ex Vivo/In Vitro Study.

Little is known of the effects of nanoparticles in human systems, let alone in diseased individuals and nanotechnology has preceded nanotoxicology. Therefore, the effects of titanium dioxide (TiO2) nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases [lung cancer, chronic obstructive pulmonary disease (COPD) and asthma] were compared with those in healthy Individuals, to determine differences in sensitivity to nanochemical insult. The Comet assay was performed according to recommended guidelines. The micronucleus assay and ras oncoprotein detection were conducted according to published standard methods. The results showed statistically significant concentration-dependent genotoxic effects of TiO2 NPs in both respiratory patient and control groups in the Comet assay. The TiO2 NPs caused DNA damage in a concentration dependent manner in both groups (respiratory and healthy controls) with the exception of the lowest TiO2 concentration (10 µg/ml) which did not induce significant damage in healthy controls (n.s). When OTM data were used to compare the whole patient group and the control group, the patient group had more DNA damage (p > 0.001) with the exception of 10 µg/ml of TiO2 that caused less significant damage to patient lymphocytes (p < 0.05). Similarly, there was an increase in the pattern of cytogenetic damage measured in the MN assay without statistical significance except when compared to the negative control of healthy individuals. Furthermore, when modulation of ras p21 expression was investigated, regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels. Results were achieved in the absence of cytotoxicity.

Cellular internalization and antioxidant activity of cerium oxide nanoparticles in human monocytic leukemia cells.

Overproduction of free radicals contributes to oxidative stress and inflammation leading to various disease conditions. Cerium oxide nanoparticles (nanoceria) have been shown to scavenge free radicals and have the potential for being used as a therapeutic agent in disease conditions. Therefore, in the present study, human monocytic leukemia cells (THP-1) were used as a model to evaluate the uptake and free radical scavenging activity of nanoceria. Our data showed a significant (P<0.05) increase in the internalization of nanoceria in a concentration-dependent (10-100 µg/mL) manner in THP-1 cells. Although no cytotoxicity was observed at these concentrations, nanoceria significantly (P<0.05) reduced the amount of reactive oxygen species. This was evident by a significant (P<0.05) decrease in the 2,7-dichlorofluorescein diacetate fluorescence observed in flow cytometry and fluorescence microscopy. The present study shows that nanoceria have therapeutic potential in diseases such as cancer.

Preferential binding of fullerene and fullerenol with the N-terminal and middle regions of amyloid beta peptide: an in silico investigation.

Amyloid beta (Aß) deposits are implicated in the pathogenesis of debilitating neurodegenerative disorders such as Alzheimer's disease. In the present study, the interactions of carbon-based nanoparticles (NPs) such as fullerene and fullerenol having different surface chemistry with Aß were investigated using molecular dynamics simulations and docking studies. A detailed analysis of docking results showed that in 68% of the Aß conformations, fullerene and fullerenol showed interactions with the N-terminal region of the peptide. However, the high-affinity binding site (E=-48.31 kJ/mol) of fullerene resides in the hydrophobic middle region of the peptide, whereas fullerenol interacts favorably with the charged N-terminal region with a binding energy of -50.42 kJ/mol. The above differences in binding could be attributed to the surface chemistry of fullerene and fullerenol. Moreover, the N-terminal and middle regions of Aß play an important role in Aß aggregation. Therefore, the binding of fullerene and fullerenol could inhibit amyloid aggregation. This information will be helpful in designing NPs for targeting amyloid-related disorders.

Curcumin Ag nanoconjugates for improved therapeutic effects in cancer.

Curcumin has a broad spectrum of pharmacological activities, one of them is anticancer activity that is mediated through multiple mechanisms. The major disadvantage associated with the use of curcumin is its low bioavailability due to its poor aqueous solubility. Nanoformulations of curcumin provide an effective solution for this problem. In this study, we have synthesized curcumin Ag nanoconjugates and evaluated their anticancer potential.

Synthesis of biocompatible iron oxide nanoparticles as a drug delivery vehicle.

Over the last decade, there has been growing interest in developing novel nanoparticles (NPs) for biomedical applications. A safe-by-design approach was used in this study to synthesize biocompatible iron oxide NPs. The size of the particles obtained was ~100 nm. Although these NPs were significantly (P<0.05) internalized in MCF-7 (human breast adenocarcinoma cell line) cells, no adverse effect was observed in the cells as assessed by cytotoxicity assays (neutral red uptake and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and cell cycle analysis. Our data demonstrate the potential of iron oxide NPs as a biocompatible carrier for targeted drug delivery.

Monitoring characteristics and genotoxic effects of engineered nanoparticle-protein corona.

Engineered nanoparticles (ENPs) possess different physical and chemical properties compared to their bulk counterparts. These unique properties have found application in various products in the area of therapeutics, consumer goods, environmental remediation, optical and electronic fields. This has also increased the likelihood of their release into the environment thereby affecting human health and ecosystem. ENPs, when in contact with the biological system have various physical and chemical interactions with cellular macromolecules including proteins. These interactions lead to the formation of protein corona around the ENPs. Consequently, living systems interact with the protein-coated ENP rather than with a bare ENP. This ENP-protein interaction influences uptake, accumulation, distribution and clearance and thereby affecting the cytotoxic and genotoxic responses. Although there are few studies which discussed the fate of ENPs, there is a need for extensive research in the field of ENPs, to understand the interaction of ENPs with biological systems for their safe and productive application.

Heteroagglomeration of zinc oxide nanoparticles with clay mineral modulates the bioavailability and toxicity of nanoparticle in Tetrahymena pyriformis.

The extensive use of zinc oxide nanoparticles (ZnO NPs) in cosmetics, sunscreens and healthcare products increases their release in the aquatic environment. The present study explored the possible interaction of ZnO NPs with montmorillonite clay minerals in aqueous conditions. An addition of ZnO NPs on clay suspension significantly (p<0.05) increases the hydrodymic size of clay particles from 1652±90nm to 2158±13nm due to heteroagglomeration. The electrokinetic measurements showed a significant (p<0.05) difference in the electrophoretic mobilities of bare (-1.80±0.03µmcm/Vs) and ZnO NPs-clay association (-1.37±0.03µmcm/Vs) that results to the electrostatic interaction between ZnO NPs and clay particles. The attenuated total reflectance Fourier transform infrared spectroscopy analysis of ZnO NPs-clay association demonstrated the binding of ZnO NPs with the Si-O-Al region on the edges of clay particles. The increase in size of ZnO NPs-clay heteroagglomerates further leads to their sedimentation at 24h. Although, the stability of ZnO NPs in the clay suspension was decreased due to heteroagglomeration, but the bioavailability and toxicity of ZnO NPs-clay heteroagglomerates in Tetrahymena pyriformis was enhanced. These observations provide an evidence on possible mechanisms available in natural environment that can facilitate nanoparticles entry into the organisms present in lower trophic levels of the food web.

Laboratory Scale Microbial Food Chain To Study Bioaccumulation, Biomagnification, and Ecotoxicity of Cadmium Telluride Quantum Dots.

The increasing applications of engineered nanomaterials (ENMs) in consumer products warrant a careful evaluation of their trophic transfer and consequent ecological impact. In the present study, a laboratory scale aquatic microbial food chain was established using bacteria (Escherichia coli (E. coli)) as a prey and ciliated protozoan (Paramecium caudatum) as a predator organism to determine the impact of cadmium telluride quantum dots (CdTe QDs). We observed that 29% of bacterivory potential of paramecium was lost, including an ∼12 h delay in doubling time on exposure to 25 mg/L CdTe QD (∼4 nm) as compared to control. The fluorescence based stoichiometric analysis revealed that 65% of the QDs bioaccumulated when paramecia were exposed to 25 mg/L QDs at 24 h. There was a significant (p < 0.05) increase in cellular cadmium (Cd) concentration at 24 h (306 ± 192 mg/L) as compared to 1 h (152 ± 50 mg/L). Moreover, the accumulation of Cd in E. coli (147 ± 25 mg/L) at 1 h of exposure to 25 mg/L QDs transferred 1.4 times higher Cd (207 ± 24 mg/L; biomagnification factor = 1.4) to its predator, paramecium.

Zinc oxide nanoparticle induced age dependent immunotoxicity in BALB/c mice.

Zinc oxide (ZnO) nanoparticles (NPs) have potential applications in cosmetics, food packaging and biomedicine but concerns regarding their safety need to be addressed. In the present study, the immunotoxic potential of ZnO NPs was evaluated in different ages of BALB/c mice after sub-acute exposure. The cytokine release, immunophenotyping, distribution of ZnO NPs and ultrastructural changes were assessed. A significant (p < 0.05) change in the CD4- and CD8-cells, levels of IL-6, IFN-γ and TNF-α and reactive oxygen species were observed in aged mice. In juvenile mice, increase in reactive oxygen species and IL-6 and TNF-α levels was observed with no significant changes in adult mice. A significant (p < 0.05) increase in the expression levels of mitogen activated protein kinase (MAPK) cascade proteins such as phospho-ERK1/2, phospho-JNK and phospho-p38 were also induced in aged mice. Collectively, our results indicate that the aged mice are more susceptible to ZnO NP induced immunotoxicity.

Montmorillonite clay alters toxicity of silver nanoparticles in zebrafish (Danio rerio) eleutheroembryo.

An exponential development in the use of silver nanoparticles (AgNPs) in consumer products has accelerated their release in aquatic environment. As the AgNPs enters into the aquatic systems, their fate may change due to interactions with abiotic (e.g. clay particles) or biotic factors. The abundantly present clay particles are expected to more prone for interaction with nanoparticles in aquatic systems. In the present study, it is demonstrated that AgNPs interacts with clay particles and forms heteroagglomerates. Furthermore, an impact on toxicity potential of AgNPs after interactions with clay particles was assessed by using zebrafish eleutheroembryos (72 h post hatching) as an in vivo model. The mortality rate of zebrafish eleutheroembryos was higher in case of exposure to AgNPs-clay complexes (pH 4.0 and 7.0) as compared to bare AgNPs. In addition, at earlier time points, the eleutheroembryos expressed higher levels of morphological changes in tail, yolk and pericardia, but the edema in yolk sac was followed by cell death. It can be concluded from the observations made in the present study that the inorganic colloids in the aquatic matrices can alter the fate and toxicity potential of nanoparticles.

Assessment of agglomeration, co-sedimentation and trophic transfer of titanium dioxide nanoparticles in a laboratory-scale predator-prey model system.

Nano titanium dioxide (nTiO2) is the most abundantly released engineered nanomaterial (ENM) in aquatic environments. Therefore, it is prudent to assess its fate and its effects on lower trophic-level organisms in the aquatic food chain. A predator-and-prey-based laboratory microcosm was established using Paramecium caudatum and Escherichia coli to evaluate the effects of nTiO2. The surface interaction of nTiO2 with E. coli significantly increased after the addition of Paramecium into the microcosm. This interaction favoured the hetero-agglomeration and co-sedimentation of nTiO2. The extent of nTiO2 agglomeration under experimental conditions was as follows: combined E. coli and Paramecium > Paramecium only > E. coli only > without E. coli or Paramecium. An increase in nTiO2 internalisation in Paramecium cells was also observed in the presence or absence of E. coli cells. These interactions and nTiO2 internalisation in Paramecium cells induced statistically significant (p < 0.05) effects on growth and the bacterial ingestion rate at 24 h. These findings provide new insights into the fate of nTiO2 in the presence of bacterial-ciliate interactions in the aquatic environment.

Cell cycle dependent cellular uptake of zinc oxide nanoparticles in human epidermal cells.

Metal oxide nanoparticles (NPs), including zinc oxide (ZnO) NPs have shown success for use as vehicles for drug delivery and targeting gene delivery in many diseases like cancer. Current anticancer chemotherapeutics fail to effectively differentiate between cancerous and normal cells. There is an urgent need to develop novel drug delivery system that can better target cancer cells while sparing normal cells and tissues. Particularly, ZnO NPs exhibit a high degree of cancer cell selectivity and induce cell death, oxidative stress, interference with the cell cycle progression and genotoxicity in cancerous cells. In this scenario, effective cellular uptake of NP seems to be crucial, which is shown to be affected by cell cycle progression. In the present study, the cytotoxic potential of ZnO NPs and the effect of different cell cycle phases on the uptake of ZnO NPs were examined in A431 cells. It is shown that the ZnO NPs led to cell death and reactive oxygen species generation and were able to induce cell cycle arrest in S and G2/M phase with the higher uptake in G2/M phase compared with other phases.