Nano and Micro Graphene & Parasites Causing Blood Clots Seen in the Blood of the VAXXed & UNVAXXed!
Updated: Feb 11, 2023
Dear Family and Friends,
Thank you for your love and support for over forty years. I am truly thankful, grateful and humbled by your generosity and your prayers.
The following are just a few examples of the thousands of micrographs I have observed and documented in the last several months viewed under Brightfield, pHase Contrast and Darkfield Microscopy, Scanning Electron Microscopy[SEM} and Transmission Electron Microscopy[TEM].
What Does Healthy Blood Look Like?
1. Healthy capillary human blood from a UNVAXXinated male – appears even in color, even in shape and even in size as observed under Brightfield, pHase Contrast and Darkfield microscopy.
This is What Healthy Blood Looks Like! What Does YOUR Blood Look Like? Keep Reading!
What Does UnHealthy Blood Look Like Full of Graphene, Iron, Aluminum, Parasites, Acid Crystals and Blood Clots?
2. The micrograph below shows a symplast or a micro clot of enlarged red blood cells or a blood clot observed using a compound pHase contrast light microscope at 1500x magnification. These micro clots are now commonly seen in the live unstained blood of humans and animals due to acidic/toxic poison now found in food, water, air, vaccines, nutritional supplements and legend drugs. The hemoglobin, the molecule of the red blood cells adsorbs and absorbs metabolic, dietary, respiratory and environmental acidic poisonous waste in order to protect the delicate pH balance of the vascular fluids at 7.365 pH and -20.5 mV and the interstitial fluids that surround all body cells at a 8.4 pH and -80 mV. The red blood cells NOT the white blood cells are the primary protectors of the body cells that make up all organs, glands and tissues. White blood cell are secondary to red blood cells in providing immunity which is a major oversight in current medical microbiology.
The symptoms of micro clots caused by acidic chemical and radiation poisoning are as follows:
Low energy
Cold hands
Cold feet
Itchy skin
Lightheadedness
Dizziness
Muddle thinking
Forgetfulness
Foggy thinking
Dry cough
Fever
Hypoxia
Hypercapnia
DIC or Pathological Blood Coagulation
Decompensated acidosis of the vascular and interstitial fluids
Death
3. A large symplast of magnetic nano graphene oxide observed in the live unchanged and unstained capillary blood using pHase Contrast Microscopy of a VAXXinated male.
Targeted Red Blood Cell Deficient in Hemoglobin with Large Symplasts of Metallic Graphene Oxide Surrounded by Blood Clots!
4. Ferric oxide with lactic and sulphuric acid crystals observed in the live capillary blood from a VAXXinated male using Brightfield, pHase contrast microscopy and confirmed with UV absorbance and Fluorescence Spectroscopy, Scanning Electron Microscopy, Transmission Electron Microscopy, Energy Dispersive Spectroscopy, X-ray Diffractometer and Nuclear Magnetic Resonance instruments.
5. A large symplast of Nano Graphene oxide particulates observed in the live unchanged and unstained capillary blood of a VAXXinated female with metastatic lung and brain cancer patient after chemotherapy.
6. A large symplast of Nano Graphene Oxide observed in the live unchanged capillary blood under pHase contrast microscopy of a UNVAXXinated female. I call these symplasts of graphene, ” Graphene Atomic Bombs”
IMMENSE HARM FROM CRYSTALLIZED BLOOD CELLS FROM GRAPHENE POISONING POST-VAXXinated
7. Graphene oxide in the center of what appears as a vulgaris bulgarus parasite at 1500x magnification observed in the live capillary blood under phase contrast microscopy in a VAXXinated female.
8. Pathological blood coagulation combined with a host of Wuchereria bancrofti, Microfilariae, Rope and Trypanosoma Cruzi blood parasites found in living capillary blood observed under pHase Contrast Microscopy. These parasites mainly live in the lymphatic vessels, but occasionally they are found in the living capillary blood and can thrive for up to ten years in animal and human blood. These parasites destroy the health of your interstitial and vadcular fluids, causing cellular and tissue acidification, inflammation and degeneration of the cell membranes and genetic matter leading to chronic disease! You pick up these parasites when eating sushi, chicken, beef, shrimp, pork and NOW through VAXXination! It is notable that shrimp, crab and lobster have a countless number of parasites that rot the body from the inside out! Flee from these highly acidic foods and inoculations and NEVER touch them with your hands!!
9. Graphene Oxide nanowires, threads, dots and bubbles observed in the live unchanged and unstained capillary blood with Brightfield and pHase Contrast microscopy in a UNVAXXinated female.
More Micro and Nanographs of Graphene Nanowires, Threads, Ribbons, Dots and Bubbles of the Vaccinated Observed in LIVE CAPPILARY BLOOD BLOOD CLOTS!
Graphene Based Nanowires are superconductor batteries used for tissue scaffolding inside the human body and for receiving and transmitting data across the Internet of Things.
GRAPHENE NANOWIRES, RIBBONS AND NANO NETWORKS IN THE BLOOD COMBINES WITH EMF 5G RADIATION CAUSES OF INJURY AND DEATH!
10. Different morphology of nano graphene oxide as wires, ribbons, tubes, dots and sheets as observed in the live unchanged and unstained capillary blood of a VAXXinated female.
11. Nano Graphene Oxide Nanowires and Threads as observed in the live unchanged and unstained capillary blood under pHase contrast microscopy from a VAXXinated female
12. Nano Graphene Oxide sheets, tubes, nanowires, threads and dots observed in the live unchanged capillary blood under pHase contrast microscopy from a VAXXinated female. Please note the red blood cell clots throughout the smear.
13. A large Nano Graphene Oxide Tubular blood clot observed in the live unchanged and unstained capillary blood under pHase contrast microscopy from a VAXXinated male.
14. A large fibrous graphene oxide micro blood clot observed in the live unchanged and unstained capillary blood of a VAXXinated female under pHase contrast microscopy.
15. A pathological capillary blood clot containing graphene oxide threads and tubes observed in the live unchanged and unstained blood of a VAXXinated female under brightfield microscopy.
Micrographs of Healthy and UnHealthy Blood Under pHase Contrast and Brightfield Microscopy
Graphene in Nano and Micro Blood Clots in the VAXXinated and UNVAXXinated Capillary Blood!
16. Self-Assembling Graphene Oxide Based Biosensors observed in the unchanged and unstained capillary blood under pHase contrast microscopy of a VAXXinated female.
https://rumble.com/v25lyd2-young-blood-solutions-to-the-pollutions-from-nano-technology.html
17. Graphene, iron and silicon have been found in the Pfizer, Moderna and Johnson and Johnson VAXXines for children 6 months to 5 years old!
Figure 1 below reveals the cytotoxic, genotoxic and magnetic toxic spectrum of the Pfizer “vaccine” nano particulates of reduced graphene oxide or graphene hydroxide, magnesium, aluminum, silicon, chloride and calcium identified under an ESEM microscope coupled with an EDS x-ray microprobe. (X axis =KeV, Y axis = Counts)
Figure 2 below reveals the cytotoxic, genotoxic and magnetic toxic spectrum of the Moderna “vaccine” nano particulates of reduced graphene oxide or graphene hydroxide, silicon, copper, titanium, selenium, potassium and cadmium, and lead identified under an ESEM microscope coupled with an EDS x-ray microprobe. (X axis =KeV, Y axis = Counts)
CLOTS, NANOWIRES, PARASITES, SYMPLASTS OF GRAPHENE IN VAXXINATED and UNVAXXINATED BLOOD of INFANTS, CHILDREN and ADULTS!
Dr. Robert – The Beatles – https://youtu.be/Tb9L3iAUhc0
Detailed reporting (second half of video) of new findings of blood clots of different sizes, nano and micro, along with nanowires, apparent parasites, symplasts and jagged shards and coils of graphene in the live blood of vaccinated children and adults taken from capillaries (which means the nano graphene in the COVID vaccines has spread everywhere through the body) revealing that all who have taken the vaccine are in great danger of harm of different kinds including paralysis, strokes, heart attacks, sudden death, unless they wake up and start detoxing at speed.
If you would like to view more blood and vaccine micrographs of the live unchanged and unstained capillary blood and my VAXXine Research please click on the following links below:
1. Pathological Blood Coagulation – https://www.drrobertyoung.com/blog/search/blood
2. SEM and TEM Reveals Nondisclosed Ingredients in ALL VAXXinies -https://www.drrobertyoung.com/post/transmission-electron-microscopy-reveals-graphene-oxide-in-cov-19-vaccines
3. Rothschild’s Planned CONvid Plandemic – https://www.drrobertyoung.com/post/proof-rothchild-s-planned-the-plandemic
4. Second Thoughts About Viruses, Vaccines and the Viral Theory – https://www.drrobertyoung.com/post/second-thoughts-about-viruses-vaccines-the-viral-infectious-hypothesis
5. Young, RO (2016) Pathological Blood Coagulation and the Mycotoxic Oxidative Stress Test (MOST). Int J Vaccines Vaccin 2(6): 00048. DOI: 10.15406/ijvv.2016.02.00048
With Great Love and Respect,
Robert O Young MSc, DSc, PhD, Naturopathic Practitioner www.drrobertyoung.com
Follow Dr. Robert O. Young on Twitter at: https://twitter.com/phmiraclelife
You can support the research of Dr. Robert O. Young with your prayers and donations at: https://www.givesendgo.com/research
https://rumble.com/v18ku7h-5g-radiation-poisoning-combined-with-graphene-poisoning-of-the-blood.html
Understand Why Blood Clots Form Inside the Blood Vessels!
Read Dr. Robert O. Young’s Peered Review Scientific Research Article Published in the International Journal of Vaccines and Vaccination on Pathological Blood Coagulation! (2016)
Pathological Blood Coagulation and the Mycotoxic Oxidative Stress Testing, Young RO (2016) Pathological Blood Coagulation and the Mycotoxic Oxidative Stress Test (MOST). Int J Vaccines Vaccin 2(6): 00048. DOI: 10.15406/ijvv.2016.02.00048
https://www.drrobertyoung.com/post/nano-and-micro-clots-seen-in-the-blood-of-the-vaxxinated-nonvaxxinated
References
[1] Young RO (2016) Pathological Blood Coagulation and the Mycotoxic Oxidative Stress Test (MOST). Int J Vaccines Vaccin 2(6): 00048. DOI: 10.15406/ijvv.2016.02.00048
[2] Young RO (2016) Second Thoughts about Viruses, Vaccines, and the HIV/AIDS Hypothesis – Part 1. Int J Vaccines Vaccin 2(3): 00032. DOI: 10.15406/ijvv.2016.02.00032
[3] Young RO (2016) Second Thoughts Concerning Viruses, Vaccines and the HIV/AIDS Hypothesis – Part 2. Int J Vaccines Vaccin 2(3): 00034. DOI: 10.15406/ijvv.2016.02.00034
[4] Young RO (2016) Second Thoughts Concerning Viruses, Vaccines and the HIV/AIDS Hypothesis – Part 3 HIV/AIDS
and the Monomorphic Disease Model. Int J Vaccines Vaccin 2(3): 00035. DOI: 10.15406/ijvv.2016.02.00035
[5] Nanowires patent:
[6] Nanowire arrays for neurotechnology and other applications ~ patent
[7] Internalization of ferromagnetic nanowires by different living cells (2010)
[8] Hydrophobic copper nanowires for enhancing condensation heat transfer
[9] Rotational Maneuver of Ferromagnetic Nanowires for Cell Manipulation
[10] Internalization of ferromagnetic nanowires by different living cells
[11] Ultrathin gold nanowires to enhance radiation therapy
Scanning Electron Microscopy (SEM) of Nano Reduced Graphene Hydroxide and Nano Gold Observed as Wires, Threads, Ribbons and Tubes Removed from the Arteries and Veins of a Deceased Cancer Patient Who Received Radiation Therapy
The Beatles Song – Dr. Robert – Drink Your Cup of Greens
NEED MORE PROOF!
Article 1
August 2021
Graphene oxide (GO) nanomaterials have significant advantages for drug delivery and electrode materials in neural science, however, their exposure risks to the central nervous system (CNS) and toxicity concerns are also increased. The current studies of GO-induced neurotoxicity remain still ambiguous, let alone the mechanism of how complicated GO chemistry affects its biological behavior with neural cells. In this study, we characterized the commercially available GO in detail and investigated its biological adverse effects using cultured SH-SY5Y cells. We found that ultrasonic processing in medium changed the oxidation status and surface reactivity on the planar surface of GO due to its hydration activity, causing lipid peroxidation and cell membrane damage. Subsequently, ROS-disrupted mitochondrial homeostasis, resulting from the activation of NOX2 signaling, was observed following GO internalization. The autophagy-lysosomal network was initiated as a defensive reaction to obliterate oxidative damaged mitochondria and foreign nanomaterials, which was ineffective due to reduced lysosomal degradation capacity. These sequential cellular responses exacerbated mitochondrial stress, leading to apoptotic cell death. These data highlight the importance of the structure-related activity of GO on its biological properties and provide an in-depth understanding of how GO-derived cellular redox signaling induces mitochondrion-related cascades that modulate cell functionality and survival.
Article 2
Graphene and its derivatives (for example, nanoscale graphene oxide (NGO)) have emerged as extremely attractive nanomaterials for a wide range of applications, including diagnostics and therapeutics. In this work, we present a systematic study on the in vivo distribution and pulmonary toxicity of NGO for up to 3 months after exposure. Radioisotope tracing and morphological observation demonstrated that intratracheally instilled NGO was mainly retained in the lung. NGO could result in acute lung injury (ALI) and chronic pulmonary fibrosis. Such NGO-induced ALI was related to oxidative stress and could effectively be relieved with dexamethasone treatment. In addition, we found that the biodistribution of 125I-NGO varied greatly from that of 125I ions, hence it is possible that nanoparticulates could deliver radioactive isotopes deep into the lung, which might settle in numerous ‘hot spots’ that could result in mutations and cancers, raising environmental concerns about the large-scale production of graphene oxide.
Article 3
April 2017
We summarized the findings of toxicity studies on graphene-based nanomaterials (GNMs) in laboratory mammals. The inhalation of graphene (GP) and graphene oxide (GO) induced only minimal pulmonary toxicity. Bolus airway exposure to GP and GO caused acute and subacute pulmonary inflammation. Large-sized GO (L-GO) was more toxic than small-sized GO (S-GO). Intratracheally administered GP passed through the air-blood barrier into the blood and intravenous GO distributed mainly in the lungs, liver, and spleen. S-GO and L-GO mainly accumulated in the liver and lungs, respectively. Limited information showed the potential behavioral, reproductive, and developmental toxicity and genotoxicity of GNMs. There are indications that oxidative stress and inflammation may be involved in the toxicity of GNMs. The surface reactivity, size, and dispersion status of GNMs play an important role in the induction of toxicity and biodistribution of GNMs. Although this review paper provides initial information on the potential toxicity of GNMs, data are still very limited, especially when taking into account the many different types of GNMs and their potential modifications. To fill the data gap, further studies should be performed using laboratory mammals exposed using the route and dose anticipated for human exposure scenarios.
Article 4
May 2014
Graphene nanoparticle dispersions show immense potential as multifunctional agents for in vivo biomedical applications. Herein, we follow regulatory guidelines for pharmaceuticals that recommend safety pharmacology assessment at least 10-100 times higher than the projected therapeutic dose, and present comprehensive single dose response, expanded acute toxicology, toxicokinetics, and respiratory/cardiovascular safety pharmacology results for intravenously administered dextran-coated graphene oxide nanoplatelet (GNP-Dex) formulations to rats at doses between 1 and 500 mg/kg. Our results indicate that the maximum tolerable dose (MTD) of GNP-Dex is between 50 mg/kg ≤ MTD < 125 mg/kg, blood half-life < 30 min, and majority of nanoparticles excreted within 24 h through feces. Histopathology changes were noted at ≥250 mg/kg in the heart, liver, lung, spleen, and kidney; we found no changes in the brain and no GNP-Dex related effects in the cardiovascular parameters or hematological factors (blood, lipid, and metabolic panels) at doses < 125 mg/kg. The results open avenues for pivotal preclinical single and repeat dose safety studies following good laboratory practices (GLP) as required by regulatory agencies for investigational new drug (IND) application
Article 5
July 2021
Nanomaterials have been widely used in many fields in the last decades, including electronics, biomedicine, cosmetics, food processing, buildings, and aeronautics. The application of these nanomaterials in the medical field could improve diagnosis, treatment, and prevention techniques. Graphene oxide (GO), an oxidized derivative of graphene, is currently used in biotechnology and medicine for cancer treatment, drug delivery, and cellular imaging. Also, GO is characterized by various physicochemical properties, including nanoscale size, high surface area, and electrical charge. However, the toxic effect of GO on living cells and organs is a limiting factor that limits its use in the medical field. Recently, numerous studies have evaluated the biocompatibility and toxicity of GO in vivo and in vitro. In general, the severity of this nanomaterial’s toxic effects varies according to the administration route, the dose to be administered, the method of GO synthesis, and its physicochemical properties. This review brings together studies on the method of synthesis and structure of GO, characterization techniques, and physicochemical properties. Also, we rely on the toxicity of GO in cellular models and biological systems. Moreover, we mention the general mechanism of its toxicity.
Article 6
July 2020
Being a member of the nanofamily, carbon nanomaterials exhibit specific properties that mostly arise from their small size. They have proved to be very promising for application in the technical and biomedical field. A wide spectrum of use implies the inevitable presence of carbon nanomaterials in the environment, thus potentially endangering their whole nature. Although scientists worldwide have conducted research investigating the impact of these materials, it is evident that there are still significant gaps concerning the knowledge of their mechanisms, as well as the prolonged and chronic exposure and effects. This manuscript summarizes the most prominent representatives of carbon nanomaterial groups, giving a brief review of their general physico-chemical properties, the most common use, and toxicity profiles. Toxicity was presented through genotoxicity and the activation of the cell signaling pathways, both including in vitro and in vivo models, mechanisms, and the consequential outcomes. Moreover, the acute toxicity of fullerenol, as one of the most commonly investigated members, was briefly presented in the final part of this review. Thinking small can greatly help us improve our lives, but also obliges us to deeply and comprehensively investigate all the possible consequences that could arise from our pure-hearted scientific ambitions and work.
Article 7
March 2016
Recently, graphene and graphene-related materials have attracted a great deal of attention due their unique physical, chemical, and biocompatibility properties and to their applications in biotechnology and medicine. However, the reports on the potential toxicity of graphene oxide (GO) in biological systems are very few. The present study investigated the response of kidneys in male Sprague-Dawley rats following exposure to 0, 10, 20 and 40 mg/Kg GO for five days. The results showed that administration of GOs significantly increased the activities of superoxide dismutase, catalase and glutathione peroxidase in a dose-dependent manner in the kidneys compared with control group. Serum creatinine and blood urea nitrogen levels were also significantly increased in rats intoxicated with GO compared with the control group. There was a significant elevation in the levels of hydrogen peroxide and lipid hydro peroxide in GOs-treated rats compared to control animals. Histopathological evaluation showed significant morphological alterations of kidneys in GO-treated rats compared to controls. Taken together, the results of this study demonstrate that GO is nephrotoxic and its toxicity may be mediated through oxidative stress. In the present work, however, we only provided preliminary information on toxicity of GO in rats; further experimental verification and mechanistic elucidation are required before GO widely used for biomedical applications.
Article 8
October 2016
Carbon-based functional nanomaterials have attracted immense scientific interest from many disciplines and, due to their extraordinary properties, have offered tremendous potential in a diverse range of applications. Among the different carbon nanomaterials, graphene is one of the newest and is considered the most important. Graphene, a monolayer material composed of sp2-hybridized carbon atoms hexagonally arranged in a two-dimensional structure, can be easily functionalized by chemical modification. Functionalized graphene and its derivatives have been used in diverse nano-biotechnological applications, such as in environmental engineering, biomedicine, and biotechnology. However, the prospective use of graphene-related materials in a biological context requires a detailed comprehension of these materials, which is essential for expanding their biomedical applications in the future. In recent years, the number of biological studies involving graphene-related nanomaterials has rapidly increased. These studies have documented the effects of the biological interactions between graphene-related materials and different organizational levels of living systems, ranging from biomolecules to animals. In the present review, we will summarize the recent progress in understanding mainly the interactions between graphene and cells. The impact of graphene on intracellular components, and especially the uptake and transport of graphene by cells, will be discussed in detail.
Article 9
July 2016
Despite graphene being proposed for a multitude of biomedical applications, there is a dearth in the fundamental cellular and molecular level understanding of how few-layer graphene (FLG) interacts with human primary cells. Herein, using human primary umbilical vein endothelial cells as model of vascular transport, we investigated the basic mechanism underlying the biological behavior of graphene. Mechanistic toxicity studies using a battery of cell based assays revealed an organized oxidative stress paradigm involving cytosolic reactive oxygen stress, mitochondrial superoxide generation, lipid peroxidation, glutathione oxidation, mitochondrial membrane depolarization, enhanced calcium efflux, all leading to cell death by apoptosis/necrosis. We further investigated the effect of graphene interactions using cDNA microarray analysis and identified potential adverse effects by down regulating key genes involved in DNA damage response and repair mechanisms. Single cell gel electrophoresis assay/Comet assay confirmed the DNA damaging potential of graphene towards human primary cells.
Article 10
December 2016
Despite the rapid expansion of the biomedical applications of graphene oxide (GO), safety issues related to GO, particularly with regard to its effects on vascular endothelial cells (ECs), have been poorly evaluated. To explore possible GO-mediated vasculature cytotoxicity and determine lateral GO size relevance, we constructed four types of GO: micrometer-sized GO (MGO; 1089.9 ± 135.3 nm), submicrometer-sized GO (SGO; 390.2 ± 51.4 nm), nanometer-sized GO (NGO; 65.5 ± 16.3 nm), and graphene quantum dots (GQDs). All types but GQD showed a significant decrease in cellular viability in a dose-dependent manner. Notably, SGO or NGO, but not MGO, potently induced apoptosis while causing no detectable necrosis. Subsequently, SGO or NGO markedly induced autophagy through a process dependent on the c-Jun N-terminal kinase (JNK)-mediated phosphorylation of B-cell lymphoma 2 (Bcl-2), leading to the dissociation of Beclin-1 from the Beclin-1–Bcl-2 complex. Autophagy suppression attenuated the SGO- or NGO-induced apoptotic cell death of ECs, suggesting that SGO- or NGO-induced cytotoxicity is associated with autophagy. Moreover, SGO or NGO significantly induced increased intracellular calcium ion (Ca2+) levels. Intracellular Ca2+chelation with BAPTA-AM significantly attenuated microtubule-associated protein 1A/1B-light chain 3-II accumulation and JNK phosphorylation, resulting in reduced autophagy. Furthermore, we found that SGO or NGO induced Ca2+ release from the endoplasmic reticulum through the PLC β3/IP3/IP3R signaling axis. These results elucidate the mechanism underlying the size-dependent cytotoxicity of GOs in the vasculature and may facilitate the development of a safer biomedical application of GOs.
Article 11
January 2018
The successful applications of graphene nanomaterials in nanobiotechnology and medicine as well as their effective translation into real clinical utility hinge significantly on a thorough understanding of their nanotoxicological profile. Of all aspects of biocompatibility, the hemocompatibility of graphene nanomaterials with different blood constituents in the circulatory system is one of the most important elements that needs to be well elucidated. Once administered into biological systems, graphene nanomaterials may inevitably come into contact with the surrounding plasma proteins and blood cells. Crucially, the interactions between these hematological entities and graphene nanomaterials will influence the overall efficacy of their biomedical applications. As such, a comprehensive understanding of the hemotoxicity of graphene nanomaterials is critically important. This review presents an up-to-date elucidation of the hemotoxicity of graphene nanomaterials through their interactions with blood proteins and cells, as well as offers some perspectives on the current challenges, opportunities, and future development of this important field.
Article 12
January 2014
The raised considerable concerns about the possible environmental health and safety impacts of graphene nanomaterials and their derivatives originated from their potential widespread applications. We performed a comprehensive study about biological interaction of grapheme nanomaterials, specifically in regard to its differential surface functionalization (oxidation status), by using OMICS in graphene oxide (GO) and reduced graphene oxide (rGO) treated HepG2 cells. Differential surface chemistry (particularly, oxidation – O/C ratio) modulates hydrophobicity/philicity of GO/rGO which in turn governs their biological interaction potentiality. Similar toxic responses (cytotoxicity, DNA damage, oxidative stress) with differential dose dependency were observed for both GO and rGO but they exhibited distinct mechanism, such as, hydrophilic GO showed cellular uptake, NADPH oxidase dependent ROS formation, high deregulation of antioxidant/DNA repair/apoptosis related genes, conversely, hydrophobic rGO was found to mostly adsorbed at cell surface without internalization, ROS generation by physical interaction, poor gene regulation etc. Global gene expression and pathway analysis displayed that TGFβ1 mediated signaling played the central role in GO induced biological/toxicological effect whereas rGO might elicited host-pathogen (viral) interaction and innate immune response through TLR4–NFkB pathway. In brief, the distinct biological and molecular mechanisms of GO/rGO were attributed to their differential surface oxidation status.
Article 13
2015
Graphene oxide (GO) is a promising nanomaterial for application in a variety of biomedical fields, including neuro-oncology, neuroimaging, neuroregeneration and drug delivery. Microglia are the central macrophage-like cells critically involved in neuroimmunity. However, the interaction between GO and microglia remained mostly unknown. The present study investigated the influence of GO on the production of proinflammatory cytokines by microglia. Primary murine microglial cells were treated with GO (1–25 μg/mL) followed by stimulation with lipopolysaccharide (LPS) for 24 h. The cell viability was measured by spectrophotometry using AlamarBlueⓇ. The levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). The IL-1β converting enzyme (ICE) activity was measured using a specific fluorescent substrate. The activity of cathepsin B and the lysosomal permeability and alkalinity were determined by flow cytometry. Treatment with GO did not affect cell viability, but significantly suppressed the production of IL-1β. In contrast, the production of TNF-α was unaltered. In addition, the lysosomal permeability and alkalinity in microglia treated with GO were increased, whereas the activity of cathepsin B and ICE was decreased. Collectively, these results demonstrated that exposure to GO differentially affected the production of proinflammatory cytokines, which is associated with the modulation of the lysosomal pathway of cytokines processing.
Article 14
January 2017
Increased production volumes and a broadening application spectrum of graphene have raised concerns about its potential adverse effects on human health. Numerous reports demonstrate that graphene irrespective of its particular form exerts its effects on a widest range of living organisms, including prokaryotic bacteria and viruses, plants, micro- and macro-invertebrates, mammalian and human cells and whole animals in vivo. However, the available experimental data is frequently a matter of significant divergence and even controversy. Therefore, we provide here a critical analysis of the most recent (2015–2016) reports accumulated in the graphene-related materials biocompatibility and toxicology field in order to elucidate the cutting edge achievements, emerging trends and future opportunities in the area. Experimental findings from the diverse in vitro and in vivo model systems are analysed in the context of the most likely graphene exposure scenarios, such as respiratory inhalation, ingestion route, parenteral administration and topical exposure through the skin. Key factors influencing the toxicity of graphene and its complex derivatives as well as potential risk mitigation approaches exploiting graphene physicochemical properties, surface modifications and possible degradation pathways are also discussed along with its emerging applications for healthcare, diagnostics and innovative therapeutic approaches.
Article 15
January 2014
Graphene and its derivatives are promising candidates for important biomedical applications because of their versatility. The prospective use of graphene-based materials in a biological context requires a detailed comprehension of the toxicity of these materials. Moreover, due to the expanding applications of nanotechnology, human and environmental exposures to graphene-based nanomaterials are likely to increase in the future. Because of the potential risk factors associated with the manufacture and use of graphene-related materials, the number of nanotoxicological studies of these compounds has been increasing rapidly in the past decade. These studies have researched the effects of the nanostructural/biological interactions on different organizational levels of the living system, from biomolecules to animals. This review discusses recent results based on in vitro and in vivo cytotoxicity and genotoxicity studies of graphene-related materials and critically examines the methodologies employed to evaluate their toxicities. The environmental impact from the manipulation and application of graphene materials is also reported and discussed. Finally, this review presents mechanistic aspects of graphene toxicity in biological systems. More detailed studies aiming to investigate the toxicity of graphene-based materials and to properly associate the biological phenomenon with their chemical, structural, and morphological variations that result from several synthetic and processing possibilities are needed. Knowledge about graphene-based materials could ensure the safe application of this versatile material. Consequently, the focus of this review is to provide a source of inspiration for new nanotoxicological approaches for graphene-based materials.
Article 16
April 2018
With the vigorous development of nanometer-sized materials, nanoproducts are becoming widely used in all aspects of life. In medicine, nanoparticles (NPs) can be used as nanoscopic drug carriers and for nanoimaging technologies. Thus, substantial attention has been paid to the potential risks of NPs. Previous studies have shown that numerous types of NPs are able to pass certain biological barriers and exert toxic effects on crucial organs, such as the brain, liver, and kidney. Only recently, attention has been directed toward the reproductive toxicity of nanomaterials. NPs can pass through the blood–testis barrier, placental barrier, and epithelial barrier, which protect reproductive tissues, and then accumulate in reproductive organs. NP accumulation damages organs (testis, epididymis, ovary, and uterus) by destroying Sertoli cells, Leydig cells, and germ cells, causing reproductive organ dysfunction that adversely affects sperm quality, quantity, morphology, and motility or reduces the number of mature oocytes and disrupts primary and secondary follicular development. In addition, NPs can disrupt the levels of secreted hormones, causing changes in sexual behavior. However, the current review primarily examines toxicological phenomena. The molecular mechanisms involved in NP toxicity to the reproductive system are not fully understood, but possible mechanisms include oxidative stress, apoptosis, inflammation, and genotoxicity. Previous studies have shown that NPs can increase inflammation, oxidative stress, and apoptosis and induce ROS, causing damage at the molecular and genetic levels which results in cytotoxicity. This review provides an understanding of the applications and toxicological effects of NPs on the reproductive system.
Article 17
2014
Concentration-dependent cyto and genotoxicities of graphene oxide (GO) and reduced GO (rGO) sheets on spermatozoa were studied. rGO sheets with various surface chemical states were achieved using hydrazine (N2H4) hydrothermal (HT) reactions and green tea polyphenols (GTPs). Although 0.1 μg mL−1 graphene could not change sperm viability and kinetic parameters, <40% and 20% of spermatozoa were viable and progressively motile, after 2 h incubation with 400 μg mL−1 GO or rGO, respectively. All the graphene nanomaterials induced concentration-dependent reductions of adenosine triphosphate and NAD+/NADH produced by spermatozoa for motility and metabolic activity. While GO, N2H4–rGO, and HT-rGO sheets caused increasing reactive oxygen species and sperm nitric oxide production, GO sheets reduced by antioxidant GTPs decreased them. Hence, physical trapping of spermatozoa by graphene (particularly GTP–rGO) is one of the important mechanisms describing the cytotoxicity, in addition to the other reactions, resulting in the inactivation and/or death of spermatozoa. Graphene genotoxicity was initiated by 1.0 μg mL−1 of N2H4–rGO and HT-rGO and 10 μg mL−1 of GO and GTP–rGO sheets. The extremely sharp edge and/or high mobility of N2H4–rGO provided easy penetration of the sheets into spermatozoa to interact with cell nuclei. In contrast, the steric effect induced by GTPs attached on rGO caused a lower genotoxicity.
Article 18
Short-term in vivo exposure to graphene oxide can cause damage to the gut and testis – ScienceDirect
April 2017
Graphene oxide (GO) has unique physicochemical properties and also has a potentially widespread use in every field of daily life (industry, science, medicine). Demand for nanotechnology is growing every year, and therefore many aspects of its toxicity and biocompatibility still require further clarification.
This research assesses the in vivo toxicity of pure and manganese ion-contaminated GO that were administrated to Acheta domesticus with food (at 200 mg kg−1 of food) throughout their ten-day adult life.
Our results showed that short-term exposure to graphene oxide in food causes an increase in the parameters of oxidative stress of the tested insects (catalase – CAT, total antioxidant capacity – TAC), induces damage to the DNA at a level of approximately 35% and contributes to a disturbance in the stages of the cell cycle and causes an increase of apoptosis. Moreover, upon analyzing histological specimens, we found numerous degenerative changes in the cells of the gut and testis of Acheta domesticus as early as ten days after applying GO.
A more complete picture of the GO risk can help to define its future applications and methods for working with the material, which may help us to avoid any adverse effects and damage to the animal.
Article 19
December 2015
In vivo dose-dependent effects of nanoscale graphene oxide (NGO) sheets on reproduction capability of Balb/C mice were investigated. Biodistribution study of the NGO sheets (intravenously injected into male mice at dose of ∼2000 μg/mL or 4 mg/kg of body weight) showed a high graphene uptake in testis. Hence, in vivo effects of the NGO sheets on important characteristics of spermatozoa (including their viability, morphology, kinetics, DNA damage and chromosomal aberration) were evaluated. Significant in vivo effects was found at the injected concentrations ≥200 μg/mL after (e.g., ∼45% reduction in sperm viability and motility at 2000 μg/mL). Observation of remarkable DNA fragmentations and chromosomal aberrations of the spermatozoa after ∼8 weeks from the first weekly injection were assigned to the involvement of the NGO in spermatogenesis of the mice. The uptake of the NGO in the testis could also increase the generation of reactive oxygen species in semen of the mice. Moreover, semen of the NGO-treated mice (containing the damaged spermatozoa) might disturb the hormone secretion and pregnant functionality of female mice (∼44, 35 and 59% reduction in fertility, gestation ability and multi-production capability) and also viability of the next generation (∼15% reduction in postnatal viability of delivered pups).
Article 20
August 2016
Carbon-based nanomaterials such as single-walled carbon nanotubes and reduced graphene oxide are currently being evaluated for biomedical applications including in vivo drug delivery and tumor imaging. Several reports have studied the toxicity of carbon nanomaterials, but their effects on human male reproduction have not been fully examined. Additionally, it is not clear whether the nanomaterial exposure has any effect on sperm sorting procedures used in clinical settings. Here, we show that the presence of functionalized single walled carbon nanotubes (SWCNT-COOH) and reduced graphene oxide at concentrations of 1–25 μg/mL do not affect sperm viability. However, SWCNT-COOH generate significant reactive superoxide species at a higher concentration (25 μg/mL), while reduced graphene oxide does not initiate reactive species in human sperm. Further, we demonstrate that exposure to these nanomaterials does not hinder the sperm sorting process, and microfluidic sorting systems can select the sperm that show low oxidative stress post-exposure.
Article 21
July 2019
Graphene, a two-dimensional carbon sheet with single-atom thickness, shows immense promise in several nanoscientific and nanotechnological applications, including in sensors, catalysis, and biomedicine. Although several studies have shown the cytotoxicity of graphene oxide in different cell types, there are no comprehensive studies on human embryonic kidney (HEK293) cells that include transcriptomic analysis and an in vitro investigation into the mechanisms of cytotoxicity following exposure to graphene oxide. Therefore, we exposed HEK293 cells to different concentrations of graphene oxide for 24 h and performed several cellular assays. Cell viability and proliferation assays revealed a significant dose-dependent cytotoxic effect on HEK293 cells. Cytotoxicity assays showed increased lactate dehydrogenase (LDH) leakage and reactive oxygen species (ROS) generation, and decreased levels of reduced glutathione (GSH) and increased level of oxidized glutathione indicative of oxidative stress. This detailed mechanistic approach showed that graphene oxide exposure elicits significant decreases in mitochondrial membrane potential and ATP synthesis, as well as in DNA damage and caspase 3 activity. Furthermore, our RNA-Seq analysis revealed that HEK293 cells exposed to graphene oxide significantly altered the expression of genes involved in multiple apoptosis-related biological pathways. Moreover, graphene oxide exposure perturbed the expression of key transcription factors, promoting these apoptosis-related pathways by regulating their downstream genes. Our analysis provides mechanistic insights into how exposure to graphene oxide induces changes in cellular responses and massive cell death in HEK293 cells. To our knowledge, this is the first study describing a combination of cellular responses and transcriptome in HEK293 cells exposed to graphene oxide nanoparticles, providing a foundation for understanding the molecular mechanisms of graphene oxide-induced cytotoxicity and for the development of new therapeutic strategies.
Article 22
May 2010
Graphitic nanomaterials such as graphene layers (G) and single-wall carbon nanotubes (SWCNT) are potential candidates in a large number of biomedical applications. However, little is known about the effects of these nanomaterials on biological systems. Here we show that the shape of these materials is directly related to their induced cellular toxicity. Both G and SWCNT induce cytotoxic effects, and these effects are concentration- and shape-dependent. Interestingly, at low concentrations, G induced stronger metabolic activity than SWCNT, a trend that reversed at higher concentrations. Lactate dehydrogenase levels were found to be significantly higher for SWCNT as compared to the G samples. Moreover, reactive oxygen species were generated in a concentration- and time-dependent manner after exposure to G, indicating an oxidative stress mechanism. Furthermore, time-dependent caspase 3 activation after exposure to G (10 μg/mL) shows evidence of apoptosis. Altogether these studies suggest different biological activities of the graphitic nanomaterials, with the shape playing a primary role.
Article 23
February 2021
Arguments regarding the biocompatibility of graphene-based materials (GBMs) have never ceased. Particularly, the genotoxicity (e.g., DNA damage) of GBMs has been considered the greatest risk to healthy cells. Detailed genotoxicity studies of GBMs are necessary and essential. Herein, we present our recent studies on the genotoxicity of most widely used GBMs such as graphene oxide (GO) and the chemically reduced graphene oxide (RGO) toward human retinal pigment epithelium (RPE) cells. The genotoxicity of GO and RGOs against ARPE-19 (a typical RPE cell line) cells was investigated using the alkaline comet assay, the expression level of phosphorylated p53 determined via Western blots, and the release level of reactive oxygen species (ROS). Our results suggested that both GO and RGOs induced ROS-dependent DNA damage. However, the DNA damage was enhanced following the reduction of the saturated C–O bonds in GO, suggesting that surface oxygen-containing groups played essential roles in the reduced genotoxicity of graphene.
Article 24
October 2016
Polyethylene glycol (PEG) coating has been frequently used to improve the pharmacokinetic behavior of nanoparticles. Studies that contribute to better unravel the effects of PEGylation on the toxicity of nanoparticle formulation are therefore highly relevant. In the present study, reduced graphene oxide (rGO) was functionalized with PEG, and its effects on key components of the blood–brain barrier, such as astrocytes and endothelial cells, were analyzed in culture and in an in vivo rat model. The in vitro studies demonstrated concentration-dependent toxicity. The highest concentration (100 μg/mL) of non-PEGylated rGO had a lower toxic influence on cell viability in primary cultures of astrocytes and rat brain endothelial cells, while PEGylated rGO induced deleterious effects and cell death. We assessed hippocampal BBB integrity in vivo by evaluating astrocyte activation and the expression of the endothelial tight and adherens junctions proteins. From 1 h to 7 days post-rGO-PEG systemic injection, a notable and progressive down-regulation of protein markers of astrocytes (GFAP, connexin-43), the endothelial tight (occludin), and adherens (β-catenin) junctions and basal lamina (laminin) were observed. The formation of intracellular reactive oxygen species demonstrated by increases in the enzymatic antioxidant system in the PEGylated rGO samples was indicative of oxidative stress-mediated damage. Under the experimental conditions and design of the present study the PEGylation of rGO did not improve interaction with components of the blood–brain barrier. In contrast, the attachment of PEG to rGO induced deleterious effects in comparison with the effects caused by non-PEGylated rGO.
Article 25
April 2018
Graphene oxide (GO) is considered a promising 2D material for biomedical applications. However, the biological health effects of GO are not yet fully understood, in particular for highly sensitive populations such as pregnant women and their unborn children. Especially the potential impact of GO on the human placenta, a transient and multifunctional organ that enables successful pregnancy, has not been investigated yet. Here we performed a mechanistic in vitro study on the placental uptake and biological effects of four non-labelled GO with varying physicochemical properties using the human trophoblast cell line BeWo. No overt cytotoxicity was observed for all GO materials after 48 h of exposure at concentrations up to 40 µg ml−1. However, exposure to GO materials induced a slight decrease in mitochondrial activity and human choriogonadotropin secretion. In addition, GO induced a transient opening of the trophoblast barrier as evidenced by a temporary increase in the translocation of sodium fluorescein, a marker molecule for passive transport. Evidence for cellular uptake of GO was found by transmission electron microscopy analysis, revealing uptake of even large micro-sized GO by BeWo cells. Although GO did not elicit major acute adverse effects on BeWo trophoblast cells, the pronounced cellular internalization as well as the potential adverse effects on hormone release and barrier integrity warrants further studies on the long-term consequences of GO on placental functionality in order to understand potential embryo-fetotoxic risks.
Article 26
July 2016
Despite graphene being proposed for a multitude of biomedical applications, there is a dearth in the fundamental cellular and molecular level understanding of how few-layer graphene FLG) interacts with human primary cells. Herein, using human primary umbilical vein endothelial cells as model of vascular transport, we investigated the basic mechanism underlying the biological behavior of graphene. Mechanistic toxicity studies using a battery of cell based assays revealed an organized oxidative stress paradigm involving cytosolic reactive oxygen stress, mitochondrial superoxide generation, lipid peroxidation, glutathione oxidation, mitochondrial membrane depolarization, enhanced calcium efflux, all leading to cell death by apoptosis/necrosis. We further investigated the effect of graphene interactions using cDNA microarray analysis and identified potential adverse effects by down regulating key genes involved in DNA damage response and repair mechanisms. Single cell gel electrophoresis assay/Comet assay confirmed the DNA damaging potential of graphene towards human primary cells.
Article 27
December 2020
Toxicological evaluation of graphene oxide (GO) has been actively pursued under the context of large-scale industrial production and the potential for clinical translation. Nevertheless, the safety of GO remains largely debated, especially due to the lack of toxicological profile in higher mammals. Here we show that blood exposure to GO under the maximum safe starting dose may cause accidental death of mammals, including non-human primates (1 in 5 Macaca fascicularis and 7 in 121 mice), while remains general amenable in others. Elevated levels of immunoglobulin E and severe lung injury were found in dead animals, suggesting the GO-induced acute anaphylactic reactions. Noticeably, we did not observe anaphylactic reactions and deaths for two other carbon nanomaterials, including single-walled carbon nanotubes and nanodiamonds. This difference might arise from the long in-vivo circulating time of two-dimensional GO materials. This study thus highlights the urgent need to evaluate the hypersensitivity risks of graphene and other nanomaterials.
Article 28
2016
Graphene quantum dots (GQDs) have attracted tremendous attention for biological applications. We report the first study on cytotoxicity and genotoxicity of GQDs to fibroblast cell lines (NIH-3T3 cells). The NIH-3T3 cells treated with GQDs at dosages over 50 μg mL−1 showed no significant cytotoxicity. However, the GQD-treated NIH-3T3 cells exhibited an increased expression of proteins (p53, Rad 51, and OGG1) related to DNA damage compared with untreated cells, indicating the DNA damage caused by GQDs. The GQD-induced release of reactive oxygen species (ROS) was demonstrated to be responsible for the observed DNA damage. These findings should have important implications for future applications of GQDs in biological systems.
Article 29
May 2019
The potential adverse effects of graphene quantum dots (GQDs) have increasingly attracted attention. Our present study revealed the genotoxic responses of rat alveolar macrophages (NR8383) to aminated graphene QDs (AG-QDs) and detected the cellular recovery after removing AG-QDs. Global gene expression analysis from RNA-sequencing showed that AG-QDs (100 μg/mL) caused significant alterations in expression of 2898 genes after exposure for 24 h. Among these, 1335 and 1563 genes were up-regulated and down-regulated, respectively. Based on the Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis, we found that most of the down-regulated genes were responsive to “cell cycle”, which correlated well with the cell cycle arrest data that AG-QDs triggered cell cycle arrest at S (synthesis) and G2/M (second gap/mitosis) phase. The percentages of cells in S and G2/M phase were increased by 4.5%, and 29.0%, respectively. In addition, the up-regulated genes related with “endocytosis” and “phagocytosis” were identified, which could regulate the internalization of AG-QDs by endocytosis and phagocytosis. After removing exposed AG-QDs and re-incubating the cells in fresh medium, the arrest of S and G2/M phase in NR8383 cells was reduced, and the cell cycle gradually recovered. This cellular recovery could be attributed to the cellular excretion of AG-QDs and the up-regulation of the DNA-repair-related genes (Rad51, Brca2, and Atm). The current work provides insights into the potential hazards of AG-QDs in transcriptional level and presented the long-term effects of AG-QDs on organisms in environment.
Article 30
October 2017
Graphene oxide (GO) has widespread concerns in the fields of biological sciences and medical applications. Currently, studies have reported that excessive GO exposure can cause cellular DNA damage through reactive oxygen species (ROS) generation. However, DNA damage mediated response of the base excision repair (BER) pathway due to GO exposure is not elucidated yet. Therefore, we exposed HEK293T cells and zebrafish embryos to different concentrations of GO for 24 h, and transcriptional profiles of BER pathway genes, DNA damage, and cell viability were analyzed both in vitro and in vivo. Moreover, the deformation of HEK293T cells before and after GO exposure was also investigated using atomic force microscopy (AFM) to identify the physical changes occurred in the cells’ structure. CCK-8 and Comet assay revealed the significant decrease in cell viability and increase in DNA damage in HEK293T cells at higher GO doses (25 and 50 μg/mL). Among the investigated genetic markers in HEK293T cells, BER pathway genes (APEX1, OGG1, CREB1, UNG) were significantly up-regulated upon exposure to higher GO dose (50 μg/mL), however, low exposure concentration (5, 25 μg/mL) failed to induce significant genetic induction except for CREB1 at 25 μg/mL. Additionally, the viscosity of HEK293T cells decreased upon GO exposure. In zebrafish, the results of up-regulated gene expressions (apex1, ogg1, polb, creb1) were consistent with those in the HEK293T cells. Taken all together, the exposure to elevated GO concentration could cause DNA damage to HEK293T cells and zebrafish embryos; BER pathway could be proposed as the possible inner response mechanism.
Article 31
April 2018
Graphene quantum dots (GQDs) have attracted significant interests due to their unique chemical and physical properties. In this study, we investigated the potential effects of hydroxyl-modified GQDs (OH-GQDs) on the human esophageal epithelial cell line HET-1A. Our data revealed significant cytotoxicity of OH-GQDs which decreased the viability of HET-1A in a dose and time-dependent manner. The moderate concentration (25 or 50 µg/ml) of OH-GQDs significantly blocked HET-1A cells in G0/G1 cell cycle phase. An increased percentage of γpAX-positive and genomically unstable cells were also detected in cells treated with different doses of OH-GQDs (25, 50, and 100 µg/ml). Microarray data revealed that OH-GQDs treatment down-regulated genes related to DNA damage repair, cell cycle regulation and cytoskeleton signal pathways indicating a novel role of OH-GQDs. Consistent with the microarray data, OH-GQDs disrupted microtubule structure and inhibited microtubule regrowth around centrosomes in HET-1A cells. In conclusion, our findings provide important evidence for considering the application of OH-GQDs in biomedical fields.
Article 32
DNA Melting and Genotoxicity Induced by Silver Nanoparticles and Graphene | Chemical Research in Toxicology (acs.org)
March 2015
We have revealed a connection between DNA-nanoparticle (NP) binding and in vitro DNA damage induced by citrate- and branched polyethylenimine-coated silver nanoparticles (c-AgNPs and b-AgNPs) as well as graphene oxide (GO) nanosheets. All three types of nanostructures triggered an early onset of DNA melting, where the extent of the melting point shift depends upon both the type and concentration of the NPs. Specifically, at a DNA/NP weight ratio of 1.1/1, the melting temperature of lambda DNA dropped from 94 °C down to 76 °C, 60 °C, and room temperature for GO, c-AgNPs and b-AgNPs, respectively. Consistently, dynamic light scattering revealed that the largest changes in DNA hydrodynamic size were also associated with the binding of b-AgNPs. Upon introduction to cells, b-AgNPs also exhibited the highest cytotoxicity, at the half-maximal inhibitory (IC50) concentrations of 3.2, 2.9, and 5.2 mg/L for B and T-lymphocyte cell lines and primary lymphocytes, compared to the values of 13.4, 12.2, and 12.5 mg/L for c-AgNPs and 331, 251, and 120 mg/L for GO nanosheets, respectively. At cytotoxic concentrations, all NPs elicited elevated genotoxicities via the increased number of micronuclei in the lymphocyte cells. However, b-AgNPs also induced micronuclei at subtoxic concentrations starting from 0.1 mg/L, likely due to their stronger cellular adhesion and internalization, as well as their subsequent interference with normal DNA synthesis or chromosome segregation during the cell cycle. This study facilitates our understanding of the effects of NP chemical composition, surface charge, and morphology on DNA stability and genotoxicity, with implications ranging from nanotoxicology to nanobiotechnology and nanomedicine.
Article 33
December 2019
Graphene-based materials (GBMs) have attracted many scientists because of their optical, thermal, mechanical and electronic properties. Their good dispersibility in different type of solvents including water, the possibility to formulate them according to desired function, and the wide surface area, which can allow various chemical modifications, expanded the use of these materials in biological systems. For these reasons, GBMs have been extensively studied in vitro and in vivo in the biomedical field. However, the toxicity and genotoxicity of GBMs must be thoroughly investigated before they can be translated into clinical settings. The main mechanism of graphene toxicity is thought to be caused by reactive oxygen species produced in cells, which in turn interact with various biomolecules including DNA. In this review we aimed to discuss different genotoxicity studies performed with GBMs with specific focus on the different cell types and conditions. By comparing and discussing such reports, scientists will be able to engineer non toxic GBMs for future preclinical and/or clinical studies. In order to allow a safer and faster transition to clinic, future studies should involve state-of-the-art technologies such as systems biology approaches or three-dimensional microfluidic systems, which can better predict the normal physiological scenario.
Article 34
March 2016
The widespread applications of graphene family nanomaterials (GFNs) raised the considerable concern over human health and environment. The cyto-genotoxic potentiality of GFNs has attracted much more attention, albeit the potential effects on the cellular epigenome remain largely unknown. The effects of GFNs on cellular genome were evaluated with single and double stranded DNA damage and DNA repair gene expressions while the effects on epigenome was accomplished by addressing the global DNA methylation and expression of DNA methylation machineries at non-cytotoxic to moderately cytotoxic doses in in vitro system. We used five different representatives of GFNs-pristine (GNP-Prist), carboxylated (GNP-COOH) and aminated (GNP-Np) graphene nanoplatelets as well as single layer (SLGO) and few layer (FLGO) graphene oxide. The order of single stranded DNA damage was observed as GNP-Prist ≥ GNP-COOH > GNP-Np ≥ FLGO > SLGO at 10 mg/L and marked dose dependency was found in SLGO. The GFNs possibly caused genotoxicity by affecting nucleotide excision repair and non-homologus end joining repair systems. Besides, dose dependent increase in global DNA methylation (hypermethylation) were observed in SLGO/FLGO exposure and conversely, GNPs treatment caused hypomethylation following the order as GNP-COOH > GNP-Np ≥ GNP-Prist. The decrements of DNA methyltransferase (DNMT3B gene) and methyl-CpG binding domain protein (MBD1) genes were probably the cause of global hypomethylation induced by GNPs. Conversely, the de novo methylation through the up-regulation of DNMT3B and MBD1 genes gave rise to the global DNA hypermethylation in SLGO/FLGO treated cells. In general, the GFNs induced genotoxicity and alterations of global DNA methylation exhibited compounds type specificity with differential physico-chemical properties. Taken together, our study suggests that the GFNs could cause more subtle changes in gene expression programming by modulating DNA methylation status and this information would be helpful for their prospective use in biomedical field.
Article 35
April 2013
Single-layer reduced graphene oxide nanoribbons (rGONRs) were obtained through an oxidative unzipping of multi-walled carbon nanotubes and a subsequent deoxygenation by hydrazine and bovine serum albumin. Human mesenchymal stem cells (hMSCs) were isolated from umbilical cord blood and used for checking the concentration- and time-dependent cyto- and geno-toxic effects of the rGONRs and reduced graphene oxide sheets (rGOSs). The cell viability assay indicated significant cytotoxic effects of 10 μg/mL rGONRs after 1 h exposure time, while the rGOSs exhibited the same cytotoxicity at concentration of 100 μg/mL after 96 h. The oxidative stress was found as the main mechanism involved in the cytotoxicity of the rGOSs which induced a slight cell membrane damage, while RNA efflux of the hMSCs indicated that neither generation of reactive oxygen species nor the significant membrane damage of the cells could explain the cell destructions induced by the rGONRs. Our results demonstrated that, the rGONRs could penetrate into the cells and cause DNA fragmentations as well as chromosomal aberrations, even at low concentration of 1.0 μg/mL after short exposure time of 1 h.
Article 36
September 2020
The induced membrane damage is a key mechanism for the cytotoxicity of graphene nanosheets (GNSs). In this research, the physical interaction of GNSs on model membranes was investigated using artificial membranes and plasma membrane vesicles. The effects of the GNSs on plasma membrane, lysosomal and mitochondrial membranes were investigated using rat basophilic leukemia (RBL2H3) cells via lactate dehydrogenase (LDH) assay, acridine orange staining and JC-1 probe, respectively. The physical interaction with model membranes was dominated by electrostatic forces, and the adhered GNSs disrupted the membrane. The degree of physical membrane disruption was quantified by the quartz crystal microbalance with dissipation (QCM-D), confirming the serious membrane disruption. The internalized GNSs were mainly distributed in the lysosomes. They caused plasma membrane leakage, increased the lysosomal membrane permeability (LMP), and depolarized the mitochondrial membrane potential (MMP). The increased cellular levels of reactive oxygen species (ROS) were also detected after GNS exposure. The combination of physical interaction and the excess ROS production damaged the plasma and organelle membranes in living RBL-2H3 cells. The lysosomal and mitochondrial dysfunction, and the oxidative stress further induced cell apoptosis. Specially, the exposure to 25 mg/L GNSs caused severest cell mortality, plasma membrane damage, ROS generation, MMP depolarization and apoptosis. The research findings provide more comprehensive information on the graphene-induced plasma and organelle membrane damage, which is important to understand and predict the cytotoxicity of carbon-based nanomaterials.
Article 37
March 2018
The collection of intrinsic properties possessed by graphene family nanomaterials (GFNs) results in their continuous exploitation for biomedical applications. The materials biomedical potential has motivated an upsurge in green preparation routes for the production of graphene like materials with limited toxicity. A number of bio-friendly reducing agents have been utilized for the preparation of chemically reduced graphene oxide (GO), and their resulting cytotoxic effects examined. However, the toxicology effects of one of the first biomolecules implemented for the reduction of GO, ascorbic acid (AA) has yet to be investigated. Herein, the toxicity of three distinct GFNs; GO, hydrazine reduced GO (H.rGO) and AA.rGO, prepared through diverse chemical routes are studied, to demonstrate the cytotoxic activity of a green reducer, in comparison to an established reduction method using hydrazine hydrate. The variation in atomic structure of GO, H.rGO and AA.rGO resulting from different synthesis techniques demonstrates the dependence of toxicity on particle shape and size. All GFNs induced high levels of alveolar cell toxicity. Interaction of AA.rGO with the A549 human lung epithelial carcinoma cell line resulted in increased leakage of lactate dehydrogenase, indicative of diminished cell membrane integrity. The uncharacteristic shape of the AA.rGO may be responsible for this proliferated release of the essential protein. The presented data therefore demonstrates that modification of synthetic processes significantly alter the biological activities of GFNs.
Article 38
May 2012
Graphene possesses unique physical and chemical properties, which have inspired a wide range of potential biomedical applications. However, little is known about the adverse effects of graphene on the human body and ecological environment. The purpose of our work is to make assessment on the toxicity of graphene oxide (GO) against human cell line (human bone marrow neuroblastoma cell line and human epithelial carcinoma cell line) and zebrafish (Danio rerio) by comparing the toxic effects of GO with its sister, multi-walled carbon nanotubes (MWNTs). The results show that GO has a moderate toxicity to organisms since it can induce minor (about 20%) cell growth inhibition and slight hatching delay of zebrafish embryos at a dosage of 50 mg/L, but did not result in significant increase of apoptosis in embryo, while MWNTs exhibit acute toxicity leading to a strong inhibition of cell proliferation and serious morphological defects in developing embryos even at relatively low concentration of 25 mg/L. The distinctive toxicity of GO and MWNTs should be ascribed to the different models of interaction between nanomaterials and organisms, which arises from the different geometric structures of nanomaterials. Collectively, our work suggests that GO does actual toxicity to organisms posing potential environmental risks and the result is also shedding light on the geometrical structure-dependent toxicity of graphitic nanomaterials.
Article 39
Graphene nanoparticles induces apoptosis in MCF-7 cells through mitochondrial damage and NF-KB pathway – IOPscience
July 2019
In the present study, the synthesis of reduced graphene oxide (rGO) was performed via 20 kHz frequency ultrasonic solution processing technique. Synthesis of rGo was confirmed by various techniques including color changes, UV–vis spectra, x-ray analysis, infrared spectrophotometry, scanning electron microscopy, and dynamic light-scattering. The cytotoxicity of rGO was examined against human breast cancer MCF-7 cells by measuring different parameters including MTT, suppression of NF-κB translocation, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, acridine orange-ethidium bromide staining, and single cell gel electrophoresis. Also, the gene expressions of Bax and Bcl-2 proteins were measured using quantitative PCR analysis as well as florescence microscopy and indicated that rGO induces cell death using apoptosis as an exclusive mechanism. Our results shows for the first time that the rGO inhibited the proliferation of MCF-7 cells, leading to programmed cell death through activation of the mitochondrial-mediated signaling pathway with the involvement of the NF-kB signalling pathway. Taken together the present data suggest that rGO can be possibly employed in the treatment of breast cancer with the function of a potent synergistic agent added to anticancer therapy protocols.
Article 40
June 2013
Recently, attempts have been made to apply graphene oxide (GO) in the field of biology and medicine, such as DNA sensing and drug delivery with some necessary modifications. Therefore, the toxicity of GO must be evaluated before it is applied further in biomedicine. In this paper, the cytotoxicity and genotoxicity of GO to human lung fibroblast (HLF) cells have been assessed with methyl thiazolyl tetrazolium (MTT), sub-G1 measurement and comet assays, and the mechanism of its toxicity has been explored. Various modifications of GO have been made to help us determine the factors which could affect the toxicity of GO. The results indicated that cytotoxicity and genotoxicity of GO to HLF cells were concentration dependent, and the genotoxicity induced by GO was more severe than the cytotoxicity to HLF cells. Oxidative stress mediated by GO might explain the reason of its toxic effect. Furthermore, the electronic charge on the surface of GO would play a very important role in the toxicity of GO to HLF cells.
Article 41
January 2019
Nanomaterials are widely used nowadays in a range of technological and biomedical fields. Graphene as a nanomaterial used in the health-care sector and in workplaces has raised some concerns about its toxicity. This study aimed to evaluate the cytotoxicity of graphene nanoparticles (GNPs) on the A549 epithelial cells of the human lung. The GNPs were synthesized from graphite by the modified Hummer method. The physicochemical characteristics of GNPs were identified by the transmission electron microscope, the scanning electron microscope, and the Brunauer–Emmett–Teller method. The hydrodynamic size of GNPs in the dispersion media was examined using the dynamic light scattering technique. The GNPs were dispersed, after which the A549 cells were cultured. Finally, the cell viability was assayed by the MTT assay. The statistical analysis of variance was used to describe the relationship between the concentration/time variables and the GNP-induced cell deaths. The probit regression model was also used to achieve toxicological indicators. The results showed that the toxicological effects of GNPs on the A549 epithelial cells of the human lung are dose- and time-dependent. The GNPs were more cytotoxic after a 72-h exposure period compared to a 24-h and 48-h exposure period. The inhibitory concentration of 50% and “no observed adverse effect concentration” were estimated to be 40,653.1 and 0.059 µg/mL, respectively. The results of this study can be helpful in developing the occupational exposure limit for GNPs and in improving occupational health programs in workplaces. However, more investigation is needed to specify the toxicological mechanisms of GNPs.
Article 42
December 2016
Graphene derivatives (GD) are currently being evaluated for technological and biomedical applications owing to their unique physico-chemical properties over other carbon allotrope such as carbon nanotubes (CNTs). But, the possible association of their properties with underlying in vitro effects have not fully examined. Here, we assessed the comparative interaction of three GD – graphene oxide (GO), thermally reduced GO (TRGO) and chemically reduced GO (CRGO), which significantly differ in their lateral size and functional groups density, with phenotypically different human lung cells; bronchial epithelial cells (BEAS-2B) and alveolar epithelial cells (A549). The cellular studies demonstrate that GD significantly ineternalize and induce oxidative stress mediated cytotoxicity in both cells. The toxicity intensity was in line with the reduced lateral size and increased functional groups revealed more toxicity potential of TRGO and GO respectively. Further, A549 cells showed more susceptibility than BEAS-2B which reflected cell type dependent differential cellular response. Molecular studies revealed that GD induced differential cell death mechanism which was efficiently prevented by their respective inhibitors. This is prior study to the best of our knowledge involving TRGO for its safety evaluation which provided invaluable information and new opportunities for GD based biomedical applications.
Article 43
October 2018
The increased mass production of graphene related materials (GRM), intended for a broad spectrum of applications, demands a thorough assessment of their potential hazard to humans and the environment. Particularly, the paramount concern has been expressed in regard to their interaction with the respiratory system in occupational exposure settings. It has been shown that GRM are easily respirable and can interact with lung cells resulting in the induction of oxidative stress or pulmonary inflammation. However, a comprehensive assessment of potential biological effects induced by GRM is currently hardly feasible to accomplish due to the lack of well-defined GRM materials and realistic exposure data. Herein, a 3D human lung model was combined with a commercial aerosolization system to study potential side effects of GRM. Two representative types of GRM were aerosolized onto the lung epithelial tissue surface. After 24 h post exposure, selected biological endpoints were evaluated, such as cell viability, morphology, barrier integrity, induction of (pro-)inflammation and oxidative stress reactions and compared with the reference material carbon black. Single exposure to all tested GRM at the two different exposure concentrations (∼300 and 1000 ng/cm2) did not initiate an observable adverse effect to the 3D lung model under acute exposure scenarios.
Article 44
Repeated exposure to aerosolized graphene oxide mediates autophagy inhibition and inflammation in a three-dimensional human airway model – ScienceDirect
March 2020
Hazard evaluation of engineered nanomaterials (ENMs) using real-world exposure scenario could provide better interpretation of toxicity end points for their use in the assessment of human safety and for their implications in many fields such as toxicology, nanomedicine, and so forth. However, most of the current studies, both in vivo and in vitro, do not reflect realistic conditions of human exposure to ENMs, due to the high doses implemented. Moreover, the use of cellular models cultured under submerged conditions limits their physiological relevance for lung exposure, where cells are primarily cultured at the air-liquid interface. Addressing such issues is even more challenging for emergent nanomaterials, such as graphene oxide (GO), for which little or no information on exposure is available. In this work, we studied the impact of repeated exposure of GO on a three-dimensional (3D) reconstruct of human bronchial tissue, using a nebulizer system focusing on short-term effects. The selected doses (reaching a maximum of ca. 20 μg/cm2 for a period of 4 weeks of exposure) were extrapolated from alveolar mass deposition values of a broader class of carbon-based nanomaterials, reflecting a full working lifetime of human exposure. Experimental results did not show strong toxic effects of GO in terms of viability and integrity of the lung tissue. However, since 2 weeks of treatment, repeated GO exposure elicited a proinflammatory response, moderate barrier impairment, and autophagosome accumulation, a process resulting from blockade of autophagy flux. Interestingly, the 3D airway model could recover such an effect by restoring autophagy flux at longer exposure (30 days). These findings indicate that prolonged exposure to GO produces a time window (during the 30 days of treatment set for this study) for which GO-mediated autophagy inhibition along with inflammation may potentially increase the susceptibility of exposed humans to pulmonary infections and/or lung diseases. This study also highlights the importance of using physiologically relevant in vitro models and doses derived from real-world exposure to obtain focused data for the assessment of human safety.
Article 45
December 2013
Graphene oxide (GO) has attracted enormous interests due to its extraordinary properties. Recent studies have confirmed the cytotoxicity of GO, we further investigate its mutagenic potential in this study. The results showed that GO interfered with DNA replication and induced mutagenesis at molecular level. GO treatments at concentrations of 10 and 100 μg/mL altered gene expression patterns at cellular level and 101 differentially expressed genes mediated DNA-damage control, cell apoptosis, cell cycle and metabolism. Intravenous injection of GO at 4 mg/kg for 5 consecutive days clearly induced formation of micronucleated polychromic erythrocytes in mice and its mutagenesis potential appeared to be comparable to cyclophosphamide, a classic mutagen. In conclusion, GO can induce mutagenesis both in vitro and in vivo, thus extra consideration is required for its biomedical applications.
Article 46
December 2018
Graphene oxide (GO) has the potential for wide applications, which necessitates an intensive investigation of its potential hazard on human and environmental health. Even if previous studies show reproductive toxicity in the nematode Caenorhabditis elegans, the mechanisms of reproductive toxicity by GO are poorly understood. To understand the underlying mechanisms of GO-induced reproductive toxicity, we investigated the interaction between GO and C. elegans using Raman spectroscopy, sperm counts produced by spermatogenesis, progeny and analyzed the fatty acid metabolism using molecular techniques. GO-characteristic Raman spectral bands measured throughout C. elegans, brood size and Hoecst staining of dissected gonads clearly showed GO accumulation in the reproductive organs, reduced progeny and low sperm counts, which are possibly direct results of the reproductive toxicity from GO exposure. Interestingly, reduced fatty acid metabolites, such as stearic, oleic, palmitoleic, and palmitic acids, were found with GO exposure. We found that GO increased intestinal fat accumulation in wild type N2, fat-5(tm420), and fat-7(wa36) mutants, whereas it decreased fat storage in the fat-6(tm331) and nhr-49(nr2041) mutants. GO exposure affected C. elegans fat accumulation and consumption, which was possibly regulated by daf-16 and nhr-80 gene activity. Also, GO exposure suppressed the survival of long-lived fat-5(tm420) mutants, whereas it increased the survival of short-lived nhr-49(nr2041) mutants. Hence, our studies collectively indicated that GO accumulation in reproductive organs, suppression of spermatogenesis, and the alteration of fatty acid metabolism play critical roles in understanding mechanisms of toxicity in C. Elegans.
Article 47
October 2018
Graphene oxide is the hot topic in biomedical and pharmaceutical research of the current decade. However, its complex interactions with human blood components complicate the transition from the promising in vitro results to clinical settings. Even though graphene oxide is made with the same atoms as our organs, tissues and cells, its bi-dimensional nature causes unique interactions with blood proteins and biological membranes and can lead to severe effects like thrombogenicity and immune cell activation. In this review, we will describe the journey of graphene oxide after injection into the bloodstream, from the initial interactions with plasma proteins to the formation of the “biomolecular corona”, and biodistribution. We will consider the link between the chemical properties of graphene oxide (and its functionalized/reduced derivatives), protein binding and in vivo response. We will also summarize data on biodistribution and toxicity in view of the current knowledge of the influence of the biomolecular corona on these processes. Our aim is to shed light on the unsolved problems regarding the graphene oxide corona to build the groundwork for the future development of drug delivery technology.
Article 48
May 2021
The nanotechnology enabled the development of nanomaterials (NMs) with a variety of industrial, biomedical, and consumer applications. However, the mechanism of action (MoA) and toxicity of NMs remain unclear, especially in the male reproductive system. Thus, this study aimed to perform a bibliometric and systematic review of the literature on the toxic effects of different types of NMs on the male reproductive system and function in mammalian models. A series of 236 articles related to the in vitro and in vivo reproductive toxicity of NMs in mammalian models were analyzed. The data concerning the bioaccumulation, experimental conditions (types of NMs, species, cell lines, exposure period, and routes of exposure), and the MoA and toxicity of NMs were summarized and discussed. Results showed that this field of research began in 2005 and has experienced an exponential increase since 2012. Revised data confirmed that the NMs have the ability to cross the blood-testis barrier and bioaccumulate in several organs of the male reproductive system, such as testis, prostate, epididymis, and seminal vesicle. A similar MoA and toxicity were observed after in vitro and in vivo exposure to NMs. The NM reproductive toxicity was mainly related to ROS production, oxidative stress, DNA damage and apoptosis. In conclusion, the NM exposure induces bioaccumulation and toxic effects on male reproductive system of mammal models, confirming its potential risk to human and environmental health. The knowledge concerning the NM reproductive toxicity contributes to safety and sustainable use of nanotechnology.
Article 49
February 2011
Graphite nanomaterials such as thermally exfoliated graphite oxide (GO) are versatile in many applications. However, little is known about its effects on biological systems. In this study we characrerized the GO using dynamic light scattering (DLS) along with the toxicological aspects related to cytotoxicity and apoptosis in normal human lung cells (BEAS-2B). A significant concentration and time dependent decrease in cell viability was observed at different concentrations (10-100 microg/ml) by the MTT assay after 24 and 48 h of exposure and significant increase of early and late apoptotic cells was observed as compared to control cells. Our study demonstrates that GO induces cytotoxicity and apoptosis in human lung cells.
Article 50
November 2018
Graphene and its derivatives are heralded as “miracle” materials with manifold applications in different sectors of society from electronics to energy storage to medicine. The increasing exploitation of graphene-based materials (GBMs) necessitates a comprehensive evaluation of the potential impact of these materials on human health and the environment. Here, we discuss synthesis and characterization of GBMs as well as human and environmental hazard assessment of GBMs using in vitro and in vivo model systems with the aim to understand the properties that underlie the biological effects of these materials; not all GBMs are alike, and it is essential that we disentangle the structure–activity relationships for this class of materials.
Article 51
November 2018
Graphene, graphene oxide, and reduced graphene oxide have been widely considered as promising candidates for industrial and biomedical applications due to their exceptionally high mechanical stiffness and strength, excellent electrical conductivity, high optical transparency, and good biocompatibility. In this article, we reviewed several techniques that are available for the synthesis of graphene-based nanomaterials, and discussed the biocompatibility and toxicity of such nanomaterials upon exposure to mammalian cells under in vitro and in vivo conditions. Various synthesis strategies have been developed for their fabrication, generating graphene nanomaterials with different chemical and physical properties. As such, their interactions with cells and organs are altered accordingly. Conflicting results relating biocompatibility and cytotoxicity induced by graphene nanomaterials have been reported in the literature. In particular, graphene nanomaterials that are used for in vitro cell culture and in vivo animal models may contain toxic chemical residuals, thereby interfering graphene-cell interactions and complicating interpretation of experimental results. Synthesized techniques, such as liquid phase exfoliation and wet chemical oxidation, often required toxic organic solvents, surfactants, strong acids, and oxidants for exfoliating graphite flakes. Those organic molecules and inorganic impurities that are retained in final graphene products can interact with biological cells and tissues, inducing toxicity or causing cell death eventually. The residual contaminants can cause a higher risk of graphene-induced toxicity in biological cells. This adverse effect may be partly responsible for the discrepancies between various studies in the literature.
Article 52
October 2016
Due to their unique physicochemical properties, graphene-family nanomaterials (GFNs) are widely used in many fields, especially in biomedical applications. Currently, many studies have investigated the biocompatibility and toxicity of GFNs in vivo and in vitro. Generally, GFNs may exert different degrees of toxicity in animals or cell models by following with different administration routes and penetrating through physiological barriers, subsequently being distributed in tissues or located in cells, eventually being excreted out of the bodies. This review collects studies on the toxic effects of GFNs in several organs and cell models. We also point out that various factors determine the toxicity of GFNs including the lateral size, surface structure, functionalization, charge, impurities, aggregations, and corona effect ect. In addition, several typical mechanisms underlying GFN toxicity have been revealed, for instance, physical destruction, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and necrosis. In these mechanisms, (toll-like receptors-) TLR-, transforming growth factor β- (TGF-β-) and tumor necrosis factor-alpha (TNF-α) dependent-pathways are involved in the signalling pathway network, and oxidative stress plays a crucial role in these pathways. In this review, we summarize the available information on regulating factors and the mechanisms of GFNs toxicity, and propose some challenges and suggestions for further investigations of GFNs, with the aim of completing the toxicology mechanisms, and providing suggestions to improve the biological safety of GFNs and facilitate their wide application.
Article 53
May 2015
With tremendous increase in development of nanotechnology, there is a developing enthusiasm toward the application of nanoparticles in diverse areas. Carbon nanotubes, fullerenes, quantum dots, dendrimers, iron oxide, silica, and gold and silver nanoparticles are frequently used in different applications such as drug delivery, ceramic materials, semiconductors, electronics, medicine, cosmetics, etc. Some of these nanoparticles have shown major toxic effects on fauna, flora, and human beings, such as inflammation, cytotoxicity, tissue ulceration, and reduction of cell viability. Single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) can induce oxidative stress and fibrosis in the lungs of rat and mice. SWCNTs can also induce oxidative stress to the nervous system in human beings. Inflammatory injury and respiratory distress can be observed due to TiO2 nanoparticles with small diameter. Nanoparticles can also pose detrimental effects on plants, such as decreased growth rate, genomic and proteomic changes, etc. Toxicity of nanoparticles arises because of their specific characteristics, such as greater “surface area to volume ratio” compared with bulk particles of the same chemistry. The objective of this review is to critically evaluate the current literature on the toxicity of nanoparticles.
Article 54
July 2019
