A USA Court Orders the CDC to Release Data Showing 18 Million Seriously Injured by the Corona VAXXXine!
What Happened to Informed Consent?
The Court Records Showed More than 18 million people were seriously injured by their first GRAPHENE-LACED COVID-19 VAXXXination from Pfizer or Moderna and had to be taken to the hospital for emergency care.
That’s according to the CDC’s own internal data, which a US Court just ordered a federal agency to release to a watchdog group.
Instead of alerting the public to the incredible dangers of these shots and completely shutting down Joe Biden’s mass vaxxxination mandates, the CDC covered up the information until it was forced to be release by a US Judge.
The CDC started a VAXXXine injury monitoring program back at the very beginning of the GRAPHENE-LACED COVID bioweapon clot shot, rollout in December of 2020.
There were many people who were eager to help, because World governments had seriously scared these folks over this so-called novel corona virus which had never been scientifically purified and isolated. In other words, the novel corona virus was never shown to exist !
https://www.drrobertyoung.com/post/transmission-electron-microscopy-reveals-graphene-oxide-in-cov-19-vaccines
Many people thought that the so-called corona virus shots were a medical miracle in late 2020. M ore than 10 million people downloaded V-safe on their smartphones , and then proceeded to get VAXXXinated with a non-disclosed graphene-laced gain-of-function bioweapon clot shot.
https://www.actascientific.com/ASMS/ASMS-06-1351.php
Ten million people is a huge sample size for a medical study. With ten million people participating in the V-safe self-reporting system, it gave us an extremely accurate statistical model to use when studying the 230 million Americans who had received at least one COVID bioweapon shot.
The CDC tracked data in the V-safe program for the first 18 months of the vaccine’s public availability, up through July of this year. But then, strangely, the CDC never published any data from V-safe. You couldn’t see the data. So, you just had to trust the CDC, which in the past had been caught lying repeatedly.
The CDC’s main webpage concerning the mRNA Corona virus bioweapon shot still says, to this very day, “COVID-19 vaccines are safe, effective and free.” That’s the very first sentence on the website.
That’s been the CDC’s position for the entire time. The VAXXXines are safe, and they cannot hurt you.
https://rumble.com/v20v82y-how-safe-and-effective-are-vaxxxines.html
The big question is why wouldn’t the CDC release the data freely instead of the court ordering the CDC to do so following a lawsuit by the Informed Consent Action Network (ICAN)?
Of the 10 million people who participated in V-safe – again, a massive sample size – 3.3 million reported Adverse Health Impacts (AHIs) immediately after their first VAXXXination which contained graphene oxide, PEG, and parasite eggs. That’s 33% or one-in-three. Of those 3.3 million people, 1.2 million reported that they were unable to perform daily activities for a time after being VAXXXinated with the so-called corona virus shot. 1.3 million reported getting so sick from the clot shot that they had to miss school or work. And, about 800,000 reported being hospitalized as a result of receiving the COVID VAXXXination!
Causes of Death: UNVAXXXinated people (2021 – 2022) Age <=65, Died > 2020
Hospital Treatments for COVID WERE the #1 KILLER!
People who died suddenly, pulmonary embolism, and turbo cancers were all very low. There are no unknown causes of death. No myocarditis deaths.
https://stevekirsch.substack.com/p/now-everyone-can-easily-prove-the
Causes of Death: VAXXXinated People (2021 – 2022) Age <=65, Died > 2020
Stop right there! This should be a showstopper!
Cancer is next. Turbo cancer is now significant compared to what it was before. Myocarditis is now a player and is killing more than COVID.
https://stevekirsch.substack.com/p/now-everyone-can-easily-prove-the
That Last Figure is the Most Troublesome!
800,000 hospitalizations out of 10 million people? That’s an 8% hospitalization rate. It means that as many as 18 million of the 230 million people who received at least one shot may have been hospitalized with a serious adverse reaction.
A study published in June of 2021 by the National Institutes of Health – where Tony Fauci works – found that the hospitalization rate from COVID-19 for the total population was 2.1%. If you are under the age of 40, the hospitalization rate from COVID-19 is just 0.4%.
For the GRAPHENE-LACED BIOWEAPON SHOTS in the USA, the Hospitalization rate has been 8% or higher!
YOU ARE 4 TIMES MORE LIKELY TO BE HOSPITALIZED BY the Graphene-laced Jibby Jabb BIOWEAPON or SO-CALLED VAXXXINE SHOTS THAN BY a so-called novel corona virus that has never been scientifically shown to exist!
https://www.drrobertyoung.com/post/there-is-no-evidence-of-the-existence-for-any-virus-isolated-by-anyone-anywhere-in-the-world
YOU ARE 20 TIMES MORE LIKELY TO BE HOSPITALIZED BY THE GRAPHENE-LACED JIBBY JABB BIOWEAPON SHOTS IF YOU ARE UNDER AGE 40 THAN BY THE CORONA VIRUS ITSELF.
You Can ead the NIH-published study HERE !
https://pubmed.ncbi.nlm.nih.gov/33729203/
ICAN has set up a website where you can finally view the CDC’s V-safe data online. The data was released on October 3, 2022, under a court order.
You can see the data for yourself HERE !
https://icandecide.org/v-safe-data/
The CDC AND OUR GOVERNMENT have been lying to the American people about the GRAPHENE-LACED BIOWEAPON CLOT SHOTS THEY CALL vaccines all this time!
Biden Ushers in Transhumanism for ALL Americans With His Executive Order
[https://www.drrobertyoung.com/post/genetic-engineering-for-writing-the-circurity-of-human-cells-for-5g-ai-super-computer-connection]
[https://www.drrobertyoung.com/post/what-is-in-the-so-called-covid-19-vaxxxines-part-1-evidence-of-a-global-crime-against-humanity]
Only when you can understand the history and the drawbacks of human beings, will you wake up and fight against tyranny!
Should The So-called VAXXXine Be Investigated For ALL of These Sudden Injuries & Deaths?
Can YOU Explain ALL of These Incidents?
https://www.drrobertyoung.com/post/should-the-jibby-jabb-be-investigated-for-all-of-these-sudden-injuries-deaths
The Viral Theory is Scientism – Radiation & the VAXXX Causes Dis-Ease, Injury and Sudden Death!
Coronavirus Means Radiation Poisoning !
https://rumble.com/v209hls-the-viral-theory-is-scientism-radiation-and-the-vaxxx-causes-dis-ease.htm
BILL GATES ADMITS TO INVESTING IN WORLD DEPOPULATION!
https://www.bitchute.com/video/TLXAhFWKx5jM/
https://rumble.com/v2483t6-neo-aristocrats-plan-to-kill-us-dr-rima-laibow-official-testimony.-georgia-.html
“How much time he gains who does not look to see what his neighbour says or does or thinks, but only at what he does himself, to make it just and holy.” MARCUS AURELIUS
LINKS FOR MORE: DR. ROBERT YOUNG:
Dr. Young’s major article reporting his and other scientists’ findings in the 4 major COVID vaccines:
https://www.drrobertyoung.com/post/transmission-electron-microscopy-reveals-graphene-oxide-in-cov-19-vaccines
PREVIOUS PODCASTS WITH DR. YOUNG WHERE HE EXPLAINS MORE ON THESE SUBJECTS:
Report 255 | Dr. Robert Young: All Disease is Outfection Not Infection–Vaccine Nano is Bioweapon!
https://www.bitchute.com/video/rdQhuY455VmK/
Newsbreak 135–BREAKING: Immense Harm from Crystallizing Blood Cells/Graphene Poisoning Post-Vaccine
https://www.bitchute.com/video/PRBfJ6U9EZvM/
Newsbreak 133: Team of Scientists Confirm Presence of Toxins Graphene, Aluminium, Cadmium Selenide, Stainless Steel, LNP-GO Capsids, Parasites, Other Toxins Variously in 4 COVID Vaccines: Pfizer, Moderna, AstraZeneca, Johnson & Johnson
https://everydayconcerned.net/2021/08/28/newsbreak-133-team-of-scientists-confirm-presence-of-toxins-graphene-aluminium-cadmium-selenide-stainless-steel-lnp-go-capsids-parasites-other-toxins-variously-in-4-covid-vaccines-pfizer-mode/
https://www.bitchute.com/video/Z2sAH0Woz38r/
For the Carnicom Disclosure Project on Magnetism, Trans-humanism, Synthetic Biology, Vaccine Poisons, Bio Surveillance, Neuromodulation, Electromagnetic Bio-Human Effects, Virus Confusions, Terrain and Blood Health and the Secret Elixir of Life –
https://www.bitchute.com/video/reT2JVKTlWmB/
Scientific Articles – www.drrobertyoung.com/blog
Watch InfoWars – VaXXine Shedding, Blood Clots & Human Technology Bio-Implants!
“When the debate is lost, slander becomes the tool of the losers” Socrates
https://banned.video/watch?id=62d35c20f4450907412002d3
Watch InfoWars – The Bio-Digital-Nano Revolution
Special guest Dr. Robert Young makes his first appearance on the show to speak with Liberty Man about his microscopy and spectroscopy findings in the gene juice injections.
https://theinfowar.tv/watch?id=628058cf36fb3975f1698d52
Dr. Young and Liberty Man talk about FDA corruption and share perspectives on the nanotechnology aspects of the shots, including topics such as RNA interference, toxicity, shedding, blood clots, and electromagnetic frequencies, and how all of this plays into the larger agenda of a digital ID social-credit prison system.
DR. YOUNG REVEALS GRAPHENE, ALUMINIUM, LNP CAPSIDS, PARASITES IN 4 VACCINES
https://www.bitchute.com/video/7BM8rMBiQ6f9/
https://www.bitchute.com/video/gKpp2LQVH8P9/
https://www.bitchute.com/video/K9SZ2AjeLY0W/
https://www.bitchute.com/video/wkiZcWbmC4Vj/
Extraordinary and riveting account of healing from terminal lung cancer and recovering from COVID symptoms on the pH Miracle Lifestyle healing program of alkaline diet, exercise, and healing foods from Dr. Robert Young, who describes the healing power of the right foods and actions to maintain the right pH balance in the interstitial fluids of the body, and encourages all to learn more about how disease is not transmitted by germs—as Virology would have it—but cultured in acidic environments, and how switching our diets and lifestyles from acidic to alkaline can transform our lives.
Transhuman Nightmare Graphene Nanowires Torturing the VaXXed and the UNVaXXed Around the World!
https://rumble.com/v1awjj1-transhuman-nightmare-graphene-nanowires-torturing-the-vaxxed-and-the-uinvax.html
Important Peer-reviewed Scientific Articles Published in the International Journal of Vaccines and Vaccinations
Published September 20th, 2016 in the International Journal of Vaccines & Vaccinations
Second Thoughts About Viruses, Vaccines and the HIV/AIDS Hypothesis
Peer-reviewed and Published June 20th, 2016 in the Journal of Vaccines and Vaccinations
[1] 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
[2] 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
[3] 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
Published in1999 by Woodland Publishing
https://phmiracleproducts.com/collections/books-audio-video
https://phmiracleproducts.com/collections/books-audio-video
Published in the Early 90’s One Sickness, One Disease, One Treatment
One Sickness, One Disease, One Treatment First Edition Published in1996 – OUT of PRINT
“There is only one sickness and one disease, the over-acidification of the blood and then tissues due to an inverted way of living, eating and thinking.” Dr. Robert O. You ng – 1996
“There is only one treatment, restore the alkaline design of the body fluids with an alkaline lifestyle and diet.” Dr. Robert O. Young – 1996
Robert O. Young MSc, DSc, PhD, Naturopathic Practitioner – www.drrobertyoung.com
[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
https://patents.google.com/patent/US8115190B2/en
[6] Nanowire arrays for neurotechnology and other applications ~ patent
https://patents.google.com/patent/WO2016112315A3/e
[7] Internalization of ferromagnetic nanowires by different living cells (2010)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592113
[8] Hydrophobic copper nanowires for enhancing condensation heat transfer
https://www.sciencedirect.com/science/article/abs/pii/S2211285517300186
[9] Rotational Maneuver of Ferromagnetic Nanowires for Cell Manipulation
https://www.academia.edu/es/43298108/Rotational_Maneuver_of_Ferromagnetic_Nanowires_for_Cell_Manipulation
[10] Internalization of ferromagnetic nanowires by different living cells
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592113
[11] Ultrathin gold nanowires to enhance radiation therapy
https://pubmed.ncbi.nlm.nih.gov/32917209/
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
[12] DARPA-Funded Efforts in the Development of Novel Brain-Computer Interface Technologies
https://www.drrobertyoung.com/post/nano-and-micro-clots-seen-in-the-blood-of-the-vaxxinated-nonvaxxinated
https://www.drrobertyoung.com/post/nano-and-micro-clots-seen-in-the-blood-of-the-vaxxinated-nonvaxxinated
Graphene oxide disrupted mitochondrial homeostasis through inducing intracellular redox deviation and autophagy-lysosomal network dysfunction in SH-SY5Y cells – ScienceDirect
Biodistribution and pulmonary toxicity of intratracheally instilled graphene oxide in mice | NPG Asia Materials (nature.com) April 2013
A review of toxicity studies on graphene-based nanomaterials in laboratory animals – ScienceDirect
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.
Dose ranging, expanded acute toxicity and safety pharmacology studies for intravenously administered functionalized graphene nanoparticle formulations. – Abstract – Europe PMC
Synthesis and Toxicity of Graphene Oxide Nanoparticles: A Literature Review of In Vitro and In Vivo Studies (hindawi.com)
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.
The Puzzling Potential of Carbon Nanomaterials: General Properties, Application, and Toxicity (nih.gov)
Toxicity Evaluation of Graphene Oxide in Kidneys of Sprague-Dawley Rats – PubMed (nih.gov)
Interactions of graphene with mammalian cells: Molecular mechanisms and biomedical insights – ScienceDirect
Cellular and molecular mechanistic insight into the DNA-damaging potential of few-layer graphene in human primary endothelial cells – ScienceDirect
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.
Graphene oxide induces apoptotic cell death in endothelial cells by activating autophagy via calcium-dependent phosphorylation of c-Jun N-terminal kinases – ScienceDirect
Understanding the hemotoxicity of graphene nanomaterials through their interactions with blood proteins and cells | SpringerLink
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.
A systems toxicology approach to the surface functionality control of graphene–cell interactions – ScienceDirect
A DIFFERENTIAL EFFECT OF GRAPHENE OXIDE ON THE PRODUCTION OF PROINFLAMMATORY CYTOKINES BY MURINE MICROGLIA | Taiwan Veterinary Journal (worldscientific.com)
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.
Graphene toxicity as a double-edged sword of risks and exploitable opportunities: a critical analysis of the most recent trends and developments – IOPscience
Assessment of the toxic potential of graphene family nanomaterials – ScienceDirect
Potential adverse effects of nanoparticles on the reproductive system | IJN (dovepress.com)
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.
Cyto and genotoxicities of graphene oxide and reduced graphene oxide sheets on spermatozoa – RSC Advances (RSC Publishing)
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.
Short-term in vivo exposure to graphene oxide can cause damage to the gut and testis – ScienceDirect
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.
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.
Dose-dependent effects of nanoscale graphene oxide on reproduction capability of mammals –
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).
Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm | Scientific Reports (nature.com)
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.
Nanomaterials | Free Full-Text | Evaluation of Graphene Oxide Induced Cellular Toxicity and Transcriptome Analysis in Human Embryonic Kidney Cells (mdpi.com)
Cytotoxicity Effects of Graphene and Single-Wall Carbon Nanotubes in Neural Phaeochromocytoma-Derived PC12 Cells | ACS Nano
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.
PEGylation of Reduced Graphene Oxide Induces Toxicity in Cells of the Blood–Brain Barrier: An in Vitro and in Vivo Study | Molecular Pharmaceutics (acs.org)
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.
Impact of graphene oxide on human placental trophoblast viability, functionality and barrier integrity – IOPscience
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.
Cellular and molecular mechanistic insight into the DNA-damaging potential of few-layer graphene in human primary endothelial cells – ScienceDirect
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.
Blood exposure to graphene oxide may cause anaphylactic death in non-human primates – ScienceDirect
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.
Can graphene quantum dots cause DNA damage in cells? – Nanoscale (RSC Publishing)
Genotoxic response and damage recovery of macrophages to graphene quantum dots – ScienceDirect
Graphene oxide nanosheets induce DNA damage and activate the base excision repair (BER) signaling pathway both in vitro and in vivo – ScienceDirect
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.
Hydroxylated-Graphene Quantum Dots Induce DNA Damage and Disrupt Microtubule Structure in Human Esophageal Epithelial Cells | Toxicological Sciences | Oxford Academic (oup.com)
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.
A closer look at the genotoxicity of graphene based materials – IOPscience
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.
Differential genotoxic and epigenotoxic effects of graphene family nanomaterials (GFNs) in human bronchial epithelial cells – ScienceDirect
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.
Genotoxicity of graphene nanoribbons in human mesenchymal stem cells – ScienceDirect
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.
Graphene nanosheets damage the lysosomal and mitochondrial membranes and induce the apoptosis of RBL-2H3 cells – ScienceDirect
An in vitro cytotoxicity assessment of graphene nanosheets on alveolar cells – ScienceDirect
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.
Toxicity of graphene oxide and multi-walled carbon nanotubes against human cells and zebrafish | SpringerLink
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.
Graphene nanoparticles induces apoptosis in MCF-7 cells through mitochondrial damage and NF-KB pathway – IOPscience
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.
Role of surface charge and oxidative stress in cytotoxicity and genotoxicity of graphene oxide towards human lung fibroblast cells – Wang – 2013 – Journal of Applied Toxicology – Wiley Online Library
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.
An assessment of the cytotoxic effects of graphene nanoparticles on the epithelial cells of the human lung – Nafiseh Nasirzadeh, Mansur Rezazadeh Azari, Yahya Rasoulzadeh, Yousef Mohammadian, 2019 (sagepub.com)
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.
Physico-chemical properties based differential toxicity of graphene oxide/reduced graphene oxide in human lung cells mediated through oxidative stress | Scientific Reports (nature.com)
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.
Single exposure to aerosolized graphene oxide and graphene nanoplatelets did not initiate an acute biological response in a 3D human lung model – ScienceDirect
Repeated exposure to aerosolized graphene oxide mediates autophagy inhibition and inflammation in a three-dimensional human airway model – ScienceDirect
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.
Graphene oxide can induce in vitro and in vivo mutagenesis | Scientific Reports (nature.com)
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.
Graphene oxide nano-bio interaction induces inhibition of spermatogenesis and disturbance of fatty acid metabolism in the nematode Caenorhabditis elegans – PubMed (nih.gov)
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.
Graphene oxide touches blood: in vivo interactions of bio-coronated 2D materials – Nanoscale Horizons (RSC Publishing)
Can nanomaterials induce reproductive toxicity in male mammals? A historical and critical review – ScienceDirect
Toxicity of graphene in normal human lung cells (BEAS-2B) – PubMed (nih.gov)
Safety Assessment of Graphene-Based Materials: Focus on Human Health and the Environment | ACS Nano
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.
Graphene Nanomaterials: Synthesis, Biocompatibility, and Cytotoxicity (nih.gov)
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.
Toxicity of graphene-family nanoparticles: a general review of the origins and mechanisms | Particle and Fibre Toxicology | Full Text (biomedcentral.com)
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.
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.
Evaluation of Graphene Oxide Induced Cellular Toxicity and Transcriptome Analysis in Human Embryonic Kidney Cells – PubMed (nih.gov)