Graphene Oxide Exposure Research Articles

Ascorbic Acid and Graphene Oxide Exposure in the Model Organism Acheta domesticus Can Change the Reproduction Potential.

The use of nanoparticles in the industry carries the risk of their release into the environment. Based on the presumption that the primary graphene oxide (GO) toxicity mechanism is reactive oxygen species production in the cell, the question arises as to whether well-known antioxidants can protect the cell or significantly reduce the effects of GO. This study focused on the possible remedial effect of vitamin C in Acheta domesticus intoxicated with GO for whole lives. The reproduction potential was measured at the level of Vitellogenin (Vg) gene expression, Vg protein expression, hatching success, and share of nutrition in the developing egg. There was no simple relationship between the Vg gene's expression and the Vg protein content. Despite fewer eggs laid in the vitamin C groups, hatching success was high, and egg composition did not differ significantly. The exceptions were GO20 and GO20 + Vit. C groups, with a shift in the lipid content in the egg. Most likely, ascorbic acid impacts the level of Vg gene expression but does not affect the production of Vg protein or the quality of eggs laid. Low GO concentration in food did not cause adverse effects, but the relationship between GO toxicity and its concentration should be investigated more thoroughly.

Dicyandiamide Applications Mitigate the Destructive Effects of Graphene Oxide on Microbial Activity, Diversity, and Composition and Nitrous Oxide Emission in Agricultural Soil.

The widespread production and utilization of graphene oxide (GO) raise concerns about its environmental release and potential ecological impacts, particularly in agricultural soil. Effective nitrogen (N) management, especially through nitrification inhibitors like dicyandiamide (DCD), might mitigate the negative effects of GO exposure on soil microbes via N biostimulation. This study quantified changes in soil physicochemical properties, nitrous oxide (N2O) emissions, microbial activity, biomass, and community after treatments with GO and DCD. The GO exposure significantly reduced bacterial 16S rRNA gene abundance and the biomass of major bacterial phyla. It also stimulated pathways linked to human diseases. However, DCD application alleviated the negative effects of GO exposure on soil bacterial biomass. While DCD application significantly reduced soil N2O emission, the GO application tended to hinder the inhibiting performance of DCD. Our findings highlight the hazards of GO exposure to soil microbes and the potential mitigation strategy with soil N management.

Transcriptomics and metabolomics analyses of graphene oxide toxicity on porcine alveolar macrophages

Graphene oxide (GO) is a type of nanomaterial widely used in tissue engineering, photocatalysis, and biomedicine. GO has been found to produce adverse effects on a broad range of cells and tissues. However, the molecular mechanisms underlying GO toxicity still remain to be explored. In this study, using porcine alveolar macrophages as a study model, we explored the toxic effects of GO and performed genome-wide detection of genes and metabolites associated with GO exposure using RNA-seq and liquid chromatograph mass spectrometer techniques. GO exposure significantly inhibited cell viability and induced apoptosis and oxidative stress in porcine alveolar macrophages. Further, GO exposure promoted cellular inflammation by upregulating the expression of pro-inflammatory cytokines (IL-6, IL-8, and IL-12). Transcriptomic analysis of GO-exposed cells revealed 424 differentially expressed genes. Functional enrichment analysis showed that the differentially expressed genes were significantly enriched in the pathways of Ribosome and oxidative phosphorylation (OXPHOS). In addition, metabolic analysis identified 203 differential metabolites, and these metabolites were significantly enriched in biosynthesis of cofactors, purine metabolism, and nucleotide metabolism. Integrative analyses of transcriptome and metabolome showed that OXPHOS was the most significantly enriched pathway and the involved genes were downregulated. This study revealed the toxic effects of GO on porcine alveolar macrophages and provided global insights to the metabolomic and transcriptomic alterations related to GO exposure. The results contributed to our understanding of the molecular mechanism of GO, and may further promote the detection of biomarkers for the prediction and control of GO toxicity.

Involvement of ahr-dependent Cyp1a detoxification activity, oxidative stress and inflammatory regulation in response to graphene oxide exposure in rainbow trout (Oncorhynchus mykiss)

Graphene oxide (GO) is a very attractive material for use in a vast number of applications. However, before its widespread use, it is important to consider potential issues related to environmental safety to support its safe application. The aim of this study was to investigate effects on fish (rainbow trout) following GO exposure. Using both an in vitro approach with the RTL W1 rainbow trout liver cell line, and in vivo exposures, following OECD TG 203, disturbances at the cellular level as well as in the gills and liver tissue of juvenile trout were assessed. In RTL W1 cells, a time and concentration-dependent loss in cell viability, specifically plasma membrane integrity and lysosomal function, was observed after 96 h of exposure to GO at concentrations ≥18.75 mg/L. Additionally, increased reactive oxygen species (ROS) levels were evidenced at concentrations ≥18.75 mg/L, and an enhancement of metabolic activity was noted with concentrations ≥4.68 mg/L. In vivo exposures to GO did not provoke mortality in rainbow trout juveniles following 96 h exposure but led to histological alterations in gills and liver tissues, induction of enzymatic detoxification activities in the liver, as well as aryl hydrocarbon receptor (ahr)–cytochrome P450 1a (cyp1a) gene expression downregulation, and upregulation of pro-inflammatory cytokines il1b and il8 at GO concentrations ≥9.89 mg/L.

The effect of graphene oxide administration on the brains of male mice: Behavioral study and assessment of oxidative stress

Graphene oxide (GO) nanoparticles are attracting growing interest in various fields, not least because of their distinct characteristics and possible uses. However, concerns about their impact on neurological health are emerging, underlining the need for in-depth studies to assess their neurotoxicity. This study examines GO exposure's neurobehavioral and biochemical effects on the central nervous system (CNS). To this end, we administered two doses of GO (2 and 5 mg/kg GO) to mice over a 46-day treatment period. We performed a battery of behavioral tests on the mice, including the open field to assess locomotor activity, the maze plus to measure anxiety, the pole test to assess balance and the rotarod to measure motor coordination. In parallel, we analyzed malondialdehyde (MDA) levels and catalase activity in the brains of mice exposed to GO nanoparticles. In addition, X-ray energy dispersive (EDX) analysis was performed to determine the molecular composition of the brain. Our observations reveal brain alterations in mice exposed to GO by intraperitoneal injection, demonstrating a dose-dependent relationship. We identified behavioral alterations in mice exposed to GO, such as increased anxiety, decreased motor coordination, reduced locomotor activity and balance disorders. These changes were dose-dependent, suggesting a correlation between the amount of GO administered and the extent of behavioral alterations. At the same time, a dose-dependent increase in malondialdehyde and catalase activity was observed, reinforcing the correlation between exposure intensity and associated biochemical responses.

Intergenerational effects of low-dose graphene oxide exposure during pregnancy and lactation on mouse neurobehavior, reproduction and development

Graphene oxide (GO), a promising biomedical nanomaterial, has attracted increasing attention regarding its potential hazards to environment and health. However, data on its multigenerational toxicity in mammals are scarce, especially regarding GO exposure during pregnancy and lactation. In this study, we investigated the effects of low-dose (0.01–1 mg/L) GO exposure during pregnancy and lactation on reproduction, development and neurobehavior in mice through a multigenerational approach. There were no significant differences in the duration of pregnancy, litter size or survival index of the offspring between the maternal mice treated with GO and those in the control group. However, a significantly altered sex ratio was observed in F1 mice exposed to 0.01 mg/L GO. Additionally, alterations in anxiety state and decreased swimming velocity were observed in maternal mice treated with 0.01 and 1 mg/L GO. The effects of GO on neurobehavior seemed to be intensified in F1 mice, as evidenced by increased stress sensitivity, altered anxiety, decreased learning and memory, and inhibited AChE activity. Although some neurobehaviors returned to normal in F2 mice, anxiety-like behavior, increased antioxidant levels and an atrophic liver were observed in the GO 1 group. Furthermore, increases in the movement velocity and distance of F2 mice were observed in GO 0.01 group. These findings provide valuable data for evaluating the transgenerational risks associated with GO exposure among mammals.

Mechanisms of surface groups regulating developmental toxicity of graphene-based nanomaterials via glycerophospholipid metabolic pathway

Surface modification of graphene-based nanomaterials (GBNs) may occur in aquatic environment and during intentional preparation. However, the influence of the surface groups on the developmental toxicity of GBNs has not been determined. In this study, we evaluated the developmental toxicity of three GBNs including GO (graphene oxide), RGO (reduced GO) and RGO-N (aminated RGO) by employing zebrafish embryos at environmentally relevant concentrations (1–100 μg/L), and the underlying metabolic mechanisms were explored. The results showed that both GO and RGO-N disturbed the development of zebrafish embryos, and the adverse effect of GO was greater than that of RGO-N. Furthermore, the oxygen-containing groups of GBNs play a more important role in inducing developmental toxicity compared to size, defects and nitrogen-containing groups. Specifically, the epoxide and hydroxyl groups of GBNs increased their intrinsic oxidative potential, promoted the generation of ROS, and caused lipid peroxidation. Moreover, a significant decrease in guanosine and abnormal metabolism of multiple glycerophospholipids were observed in all three GBN-treated groups. Nevertheless, GO exposure triggered more metabolic activities related to lipid peroxidation than RGO or RGO-N exposure, and the disturbance intensity of the same metabolite was greater than that of the other two agents. These findings reveal underlying metabolic mechanisms of GBN-induced developmental toxicity.

Physiological and transcriptomic responses of silkworms to graphene oxide exposure

The growing use of nanomaterials has sparked significant interest in assessing the insect toxicities of nanoparticles. The silkworm, as an economically important insect, serves as a promising model for studying how insects respond to harmful substances. Here, we conducted a comprehensive investigation on the impact of graphene oxide (GO) on silkworms using a combination of physiological and transcriptome analyses. GO can enter the midguts and posterior silk glands of silkworms. High GO concentrations (> 25 mg/L) significantly (P < 0.01) inhibited larval growth. Additionally, GO (> 5 mg/L) significantly reduced the cocooning rate, and GO (> 15 mg/L) hindered oviduct development and egg laying in silkworms. GO increased the reactive oxygen species content and regulated catalase activity, suggesting that it may affect insect growth by regulating reactive oxygen detoxification. The transcriptome data analysis showed that 35 metabolism-related genes and 20 ribosome biogenesis-related genes were differentially expressed in response to GO, and their expression levels were highly correlated. Finally, we propose that a Ribosome biogenesis–Metabolic signaling network is involved in responses to GO. The research provides a new perspective on the molecular responses of insects to GO.

Non-filamentous bulking of activated sludge induced by graphene oxide: Insights from extracellular polymeric substances

Widespread use of nanomaterials raises concerns. The underlying mechanism by which graphene oxide (GO) nanoparticles causes poor settleability of activated sludge remains unclear. To explore this mechanism, three reactors with different GO concentrations were established. Extended Derjaguin-Landau-Verwey-Overbeek theory indicated that GO destroyed the property of extracellular polymeric substances (EPS), increasing the energy barrier between bacteria. Low levels of uronic acid and hydrogen bonding in exopolysaccharide weakened the EPS gelation increasing aggregation repulsion. Lager amounts of hydrophilic amino acid and looser structure of extracellular proteins for exposing inner hydrophilic groups significantly contributed to the hydrophilicity of EPS. Both changes implied deterioration in EPS structure under GO stress. Metagenome demonstrated a decrease in genes responsible for capsular polysaccharide colonization and genes regulated the translocation of loose proteins were increased, which increased repulsion between bacteria. This study elucidated that changes in EPS secretion under GO exposure are the underlying causes of poor settleability.

Short term biodistribution and in vivo toxicity assessment of intravenously injected pristine graphene oxide nanoflakes in SD rats.

The present study aimed to elucidate the short term biodistribution of nano sized graphene oxide (GO) along with the toxicological assessment under in-vivo condition with an intent to analyse the toxic effects of sudden accidental exposure of GO The synthesised GO was characterized using UV-Visible spectroscopy, XRD, FTIR, Raman spectroscopy, TGA and DLS. The morphological imaging was performed using SEM, TEM and AFM. With a lateral size of less than 300nm, these nanoparticles exhibit significant organ barrier permeability of up to 20%. Upon acute exposure to 10mg/kg dose of ICG-tagged GO nanoflakes through intravenous route, various organs such as kidney, spleen and liver were observed, and the nanoparticles predominantly accumulated in the liver upon 24h of exposure. Upon confirming the accumulation of these particles in liver through IVIS imaging, our next attempt was to analyse various biochemical and serum parameters. An elevation in various serum parameters such as ALT, AST, Creatinine and Bilirubin was observed. Similarly, in the case of biochemical parameters tested in liver homogenates, an increase in NO, Catalase, GSH, SOD, ROS, LPO, GR, GPx, and GST was observed. This study highlights the potential toxicological risk associated with GO exposure which must be taken into account for any risk analysis associated with GO based consumer products and the occupational hazards.

Long-Term Exposure of Graphene Oxide Suspension to Air Leading to Spontaneous Radical-Driven Degradation.

Understanding the environmental transformation and fate of graphene oxide (GO) is critical to estimate its engineering applications and ecological risks. While there have been numerous investigations on the physicochemical stability of GO in prolonged air-exposed solution, the potential generation of reactive radicals and their impact on the structure of GO remain unexplored. In this study, using liquid-PeakForce-mode atomic force microscopy and quadrupole time-of-flight mass spectroscopy, we report that prolonged exposure of GO to the solution leads to the generation of nanopores in the 2D network and may even cause the disintegration of its bulk structure into fragment molecules. These fragments can assemble themselves into films with the same height as the GO at the interface. Further mediated electrochemical analysis supports that the electron-donating active components of GO facilitate the conversion of O2 to •O2- radicals on the GO surface, which are subsequently converted to H2O2, ultimately leading to the formation of •OH. We experimentally confirmed that attacks from •OH radicals can break down the C-C bond network of GO, resulting in the degradation of GO into small fragment molecules. Our findings suggest that GO can exhibit chemical instability when released into aqueous solutions for prolonged periods of time, undergoing transformation into fragment molecules through self-generated •OH radicals. This finding not only sheds light on the distinctive fate of GO-based nanomaterials but also offers a guideline for their engineering applications as advanced materials.

LP-54: Ascorbic acid and graphene oxide exposure in the model organism Acheta domesticus can change the reproduction potential

LP-54: Ascorbic acid and graphene oxide exposure in the model organism Acheta domesticus can change the reproduction potential

Graphene oxide had adverse effects on sperm motility and morphology through oxidative stress

Graphene oxide (GO) is a new type of graphene material, but its effects on the male reproductive system are unclear. Here, we investigated the effects of GO on human sperm in vitro. Sperms were incubated with various doses of GO (0, 10, 20, or 40 μg/mL) for different times (1, 3, or 6 h) at 37 °C, followed by analyses of the sperm motility, viability, abnormalities, and DNA fragmentations. GO exposure significantly decreased sperm motility and viability, increased sperm abnormalities, and DNA fragmentation. Moreover, GO exposure resulted in a significant reduction of sperm mitochondrial membrane potential (MMP), which was confirmed by the ultrastructural changes of chromatin and mitochondria caused by GO. These data revealed the adverse effects of GO on sperm. Further research showed that GO exposure led to a significant increase in malondialdehyde (MDA) and reactive oxygen species (ROS) in sperm cells and a significant decrease in total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px). In addition, western blot analysis showed that the levels of Nrf-2 and HO-1 protein expression in GO-treated sperm cells were significantly increased compared to the control. These results indicated that GO had adverse effects on human sperm through oxidative stress, which was associated with Nrf-2/HO-1 signaling pathway.

Exposure of zebrafish (Danio rerio) to graphene oxide for 6 months suppressed NOD-like receptor-regulated anti-virus signaling pathways.

Environmental exposure to graphene oxide (GO) is likely to happen due to the use and disposal of these materials. Although GO-induced ecological toxicity has been evaluated before by using aquatic models such as zebrafish, previous studies typically focused on the short-term toxicity, whereas this study aimed to investigate the long-term toxicity. To this end, we exposed zebrafish to GO for 6 months, and used RNA-sequencing to reveal the changes of signaling pathways. While GO exposure showed no significant effects on locomotor activities, it induced histological changes in livers. RNA-sequencing data showed that GO altered gene expression profiles, resulting in 82 up-regulated and 275 down-regulated genes, respectively. Through the analysis of gene ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we found that GO suppressed the signaling pathways related with immune systems. We further verified that GO exposure suppressed the expression of genes involved in anti-virus responses possibly through the inhibition of genes involved in NOD-like receptor signaling pathway. Furthermore, NOD-like receptor-regulated lipid genes were also inhibited, which may consequently lead to decreased lipid staining in fish muscles. We concluded that 6 month-exposure to GO suppressed NOD-like receptor-regulated anti-virus signaling pathways in zebrafish.

Low-dose graphene oxide promotes tumor cells proliferation by activating PI3K-AKT-mTOR signaling via cellular membrane protein integrin αV

Along with the increasing production and application of graphene oxide (GO), its environmental health and safety (EHS) risks have become a global concern. Numerous studies have investigated the biosafety and toxicity mechanisms associated with GO, however, the majority of previous studies were based on its direct toxic dose, which could not reflect the realistic state of environmental exposure of GO with an indirect toxic dose (low dose). Meanwhile, the effects of low-dose GO on the progression of tumors are still unclearly. Herein, we found that GO can promote multiple types of tumor cell proliferation under its low-dose treatment. Moreover, the lateral size of GO has no obvious distinction on its promoting effect on tumor proliferation. The mechanistic investigation revealed that low-dose GO treatment increased the expression level of integrin αV protein, a cell membrane receptor, and further lead to the constitutively activated PI3K/AKT/mTOR signaling pathway and promoted mitotic progression. Collectively, these findings increased our understanding of the detrimental effects of GO in promoting tumor proliferation, as well as improved our biosafety assessment at its realistic exposure doses.

59 First-in-Human Controlled Exposure to Inhaled Graphene Oxide

Abstract Graphene oxide nanomaterials have been developed for wide-ranging applications, but has potential safety concerns for human health. Controlled inhalation exposures in human volunteers have been a vital means to determine the effects and mechanisms of ultrafine particles in air pollution, however, few studies have used this approach to explore the effects of nanomaterials. We conducted a double-blind randomised controlled study to determine whether inhalation of graphene oxide affects pulmonary or cardiovascular function. A high purity graphene oxide was synthesised with a thickness of 1-2 layers in two sizes: ‘small’ (lateral dimensions: 100-1700 nm) and ‘ultrasmall’ (30-500 nm). Graphene oxide particles at 200 µg/m3, or filtered air, were inhaled for 2 hours by 14 young healthy volunteers on repeated visits, with measurement of cardiorespiratory parameters before and across 4 hours after exposure. Graphene oxide exposure was well-tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected by graphene oxide irrespective of particle size. GO did not change blood biomarkers of coagulation, however, there was a mild increase in thrombus formation in an ex vivo model of arterial injury. Proteomics revealed very few differential plasma proteins. Overall, acute inhalation of graphene oxide was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures for risk assessment of graphene nanomaterials.

73 Evaluating the Risks of Prolonged Exposure to Graphene Oxide on Healthy and Streptococcus Pneumoniae Infected 3D Reconstituted Human Lung Cultures

Abstract The growing production of graphene oxide (GO) and GO-based products has led to an increased risk of human exposure and caused scientific and public concern about human health and safety. Even though human exposure to GO can occur via different pathways, the main exposure route for airborn GO is through the lungs, thus placing the respiratory tract at particular risk. Occupational exposure to GO might occur repeatedly over a prolonged period of time but insights on lung toxicity of long-term repeated GO administration is largely lacking. Moreover, there is a risk that GO co-exposure with prevalent lung pathogens (e.g. Streptococcus pneumoniae (SP)) could increase the susceptibility to lung infections. In this study, we aimed to investigate the impact of repeated and prolonged GO exposure in healthy and SP-infected lungs using well-characterized materials with distinct properties and reconstituted primary human bronchial epithelial cultures. Our results revealed that GO had no major toxicity effects on healthy airway epithelium even upon prolonged exposure, but alterations in inflammatory/immune signaling was apparent and showed a correlation with the lateral particle size. GO pre-exposure slightly altered the response of the airway epithelium towards subsequent SP infection as evidenced by a decreased mitochondrial activity, increased bacterial translocation and altered inflammatory/immune responses. Whole genome transcriptomic profiling is ongoing to gain insights in deregulated genes and pathways following chronic GO administration. Our findings on the health risks of GO inhalation in healthy and infected human lung is important to risk assessment and management of this promising material.

Graphene oxide exposure alters gut microbial community composition and metabolism in an in vitro human model

Graphene oxide (GO) nanomaterials have unique physicochemical properties that make them highly promising for biomedical, environmental, and agricultural applications.There is growing interest in the use of GO and extensive in vitro and in vivo studies have been conducted to assess its nanotoxicity. Although it is known that GO can alter the composition of the gut microbiota in mice and zebrafish, studies on the potential impacts of GO on the human gut microbiome are largely lacking. This study addresses an important knowledge gap by investigating the impact of GO exposure- at low (25 mg/L) and high (250 mg/L) doses under both fed (nutrient rich) and fasted (nutrient deplete) conditions- on the gut microbial communitys' structure and function, using an in vitro model. This model includes simulated oral, gastric, small intestinal phase digestion of GO followed by incubation in a colon bioreactor. 16S rRNA amplicon sequencing revealed that GO exposure resulted in a restructuring of community composition. 25 mg/L GO induced a marked decrease in the Bacteroidota phylum and increased the ratio of Firmicutes to Bacteroidota (F/B). Untargeted metabolomics on the supernatants indicated that 25 mg/L GO impaired microbial utilization and metabolism of substrates (amino acids, carbohydrate metabolites) and reduced production of beneficial microbial metabolites such as 5-hydroxyindole-3-acetic acid and GABA. Exposure to 250 mg/L GO resulted in community composition and metabolome profiles that were very similar to the controls that lacked both GO and digestive enzymes. Differential abundance analyses revealed that 3 genera from the phylum Bacteroidota (Bacteroides, Dysgonomonas, and Parabacteroides) were more abundant after 250 mg/L GO exposure, irrespective of feed state. Integrative correlation network analysis indicated that the phylum Bacteroidota showed strong positive correlations to multiple microbial metabolites including GABA and 3-indoleacetic acid, are much larger number of correlations compared to other phyla. These results show that GO exposure has a significant impact on gut microbial community composition and metabolism at both low and high GO concentrations.

Impact of Environmental Stresses on the Antibacterial Activity of Graphene Oxide (GO) Nanoparticles against P. putida Biofilms.

As the production of graphene-based nanomaterials such as GO is increasing, it is expected that a large amount of GO waste will be generated. The environment (i.e., soil and aquatic systems) will be amongst the final repositories of these wastes which means important natural microbial communities in such environments will be at risk of GO exposure. However, little is known about how these communities respond to environmental stresses in synergy with the presence of GO. In this study, the effect of three different stress conditions: temperature (5, 25 and 40 °C); pH (5 to 9) and osmotic stress (51, 219 and 320 mM NaCl) in addition to GO treatment was investigated on the viability and physiology of biofilms and planktonic cells of soil bacterium P. putida. It was found that planktonic cells were more resistant to GO alone compared to biofilms. However, the cells were sensitive to GO when exposed to pH or osmotic stresses. Temperature was not found to influence the survival of biofilm with or without exposure to GO. However, low pH caused a reduction in colony-forming units (CFU) at pHs 5 and 6 for the pre-treated samples, while biofilms at pH 7-9 did not show any decrease. Interestingly, the post-treatment of planktonic cells or biofilms with GO showed a significant reduction in CFU at all pH ranges. The effect of higher osmotic stress in combination with GO resulted in a significant reduction in biofilms. These results show that the effect of stresses naturally occurring in the environment can be affected and changed when in combination with GO and can potentially affect the balance of natural biofilms.

Antimicrobial activities of graphene oxide against biofilm and intracellular Staphylococcus aureus isolated from bovine mastitis

BackgroundS. aureus is one of the causative agents of bovine mastitis. The treatment using conventional antimicrobials has been hampered due to the development of antimicrobial resistance and the ability of the bacteria to form biofilms and localize inside the host cells.ObjectivesHere, the efficacy of graphene oxide (GO), a carbon-based nanomaterial, was tested against the biofilms and intracellular S. aureus invitro. Following that, the mechanism for the intracellular antimicrobial activities and GO toxicities was elucidated.MethodsGO antibiofilm properties were evaluated based on the disruption of biofilm structure, and the intracellular antimicrobial activities were determined by the survival of S. aureus in infected bovine mammary cells following GO exposure. The mechanism for GO intracellular antimicrobial activities was investigated using endocytosis inhibitors. GO toxicity towards the host cells was assessed using a resazurin assay.ResultsAt 100 ug/mL, GO reduced between 30 and 70% of S. aureus biofilm mass, suggesting GO’s ability to disrupt the biofilm structure. At 200 ug/mL, GO killed almost 80% of intracellular S. aureus, and the antimicrobial activities were inhibited when cells were pre-treated with cytochalasin D, suggesting GO intracellular antimicrobial activities were dependent on the actin-polymerization of the cell membrane. At < 250 ug/mL, GO enhanced the viability of the Mac-T cell, and cells were only affected at higher dosages.ConclusionThe in vitro efficacy of GO against S. aureus in vitro suggested the compound could be further tested in Vivo to zrecognize its potential as one of the components of bovine mastitis therapy.