Ex Vivo Human Colon Tissue Exposure to Pristine Graphene Activates Genes Involved in the Binding, Adhesion and Proliferation of Epithelial Cells.

Toxicology studies on pristine graphene are limited and lack significant correlations with actual human response. The goal of the current study was to determine the response of total colonic human tissue to pristine graphene exposure. Biopsy punches of colon tissues from healthy human were used to assess the biological response after ex vivo exposure to graphene at three different concentrations (1, 10, and 100 µg/mL). mRNA expression of specific genes or intestinal cytokine abundance was assessed using real-time PCR or multiplex immunoassays, respectively. Pristine graphene-activated genes that are related to binding and adhesion (GTPase and KRAS) within 2 h of exposure. Furthermore, the PCNA (proliferating cell nuclear antigen) gene was upregulated after exposure to graphene at all concentrations. Ingenuity pathway analysis revealed that STAT3 and VEGF signaling pathways (known to be involved in cell proliferation and growth) were upregulated. Graphene exposure (10 µg/mL) for 24 h significantly increased levels of pro-inflammatory cytokines IFNγ, IL-8, IL-17, IL-6, IL-9, MIP-1α, and Eotaxin. Collectively, these results indicated that graphene may activate the STAT3-IL23-IL17 response axis. The findings in this study provide information on toxicity evaluation using a human-relevant ex vivo colon model and serve as a basis for further exploration of its bio-applications.

Differential Toxicological Outcome of Corn Oil Exposure in Rats and Mice as Assessed by Microbial Composition, Epithelial Permeability, and Ileal Mucosa-Associated Immune Status.

Studies to evaluate the toxicity of xenobiotics on the human gut microbiome and related health effects require a diligent selection of (1) an appropriate animal model to facilitate toxicity assessment in predicting human exposure, and (2) an appropriate non-interfering vehicle for the administration of water insoluble compounds. In biomedical studies with water insoluble xenobiotics, corn oil is one of the most commonly used nonaqueous vehicles. This study evaluated the suitability of corn oil as a vehicle in adult female Sprague Dawley rats and adult CD-1 mice; the rodent models that are often utilized in toxicological studies. We studied the host response in terms of change in the intestinal microbiome and mRNA expression of intestinal permeability and immune response-related genes when water (control) and corn oil (2 ml/kg) were administered as a vehicle through oral gavage. The results showed that the use of corn oil as a vehicle has no adverse impact in rats for either the immune response or the intestinal microbial population. On the other hand, mice treated with corn oil showed changes in bacterial community adhered to the ileum, as well as changes in the mRNA expression of intestinal permeability-related and ileal mucosa-associated immune response genes. Overall, results of this study suggest that the type of rodent species and vehicle used in toxicological risk assessments of xenobiotics studies should be taken into consideration in the experimental setup and study design.

Impact of Pristine Graphene on Intestinal Microbiota Assessed Using a Bioreactor-Rotary Cell Culture System.

The increased use of graphene in consumer products such as food contact materials requires a thorough understanding of its effects on the gastrointestinal commensal bacterial population. During the first phase of study, three representative commensal bacterial species (L. acidophilus, B. longum, and E. coli) were exposed to different concentrations (1, 10, and 100 µg/mL) of pristine graphene for 3, 6, and 24 h in the Bioreactor Rotary Cell Culture System (BRCCS) which allowed a continuous interaction of intestinal microbiota with the pristine graphene without precipitation of test material. The results showed that pristine graphene had dose-dependent effects on the growth of selective bacteria. To study the interaction of graphene with more diverse consortia of intestinal microbiota, fresh fecal samples from laboratory rats were used. Rat fecal slurry (3%) was maintained in an anaerobic environment and treated with different concentrations (1, 10, and 100 µg/mL) of pristine graphene for 3, 6, and 24 h. Counts of viable aerobic and anaerobic bacteria were assessed and fecal slurries were also collected for microbial population shift analysis using quantitative real-time PCR, as well as 16s rRNA sequencing. The results showed a significant two-fold increase in both aerobic and anaerobic bacterial counts (expressed as colony forming unit; CFU) during the first 3 h of exposure to all pristine graphene concentrations. However, 24 h of continuous exposure resulted in a 120% decrease in the CFU of aerobic bacteria at the highest concentration and the anaerobic bacteria CFU remained unchanged. Multivariate analysis of the q-PCR data showed that the exposure time, as well as the graphene concentrations, impacted the bacterial population abundance. Community analysis of graphene-treated fecal samples by 16S sequencing revealed significant alteration of 15 taxonomic groups, including 9 species. The increased abundance of butyrate-producing bacteria (Clostridium fimetarium, Clostridium hylemona, and Sutterella wadsworthensis) was correlated with an increase of the short-chain fatty acid, butyric acid after exposure to graphene. These results clearly indicate that graphene may cause adverse effects on the intestinal microbiome at the doses equal to 100 µg/mL. Further experiments using ex vivo intestinal explants (nonanimal model) could reveal the mechanisms by which graphene could perturb the microbe-host intestinal mucosa homeostasis.

The impact of tomato fruits containing multi-walled carbon nanotube residues on human intestinal epithelial cell barrier function and intestinal microbiome composition.

Carbon nanomaterials (CNMs) can positively regulate seed germination and enhance plant growth. However, clarification of the impact of plant organs containing absorbed CNMs on animal and human health is a critical step of risk assessment for new nano-agro-technology. In this study, we have taken a comprehensive approach to studying the effect tomato fruits derived from plants exposed to multi-walled carbon nanotubes (CNTs) have on gastrointestinal epithelial barrier integrity and their impact on the human commensal intestinal microbiota using an in vitro cell culture and batch human fecal suspension models. The effects of CNTs on selected pure cultures of Salmonella enterica Typhimurium and Lactobacillus acidophilus were also evaluated. This study demonstrated that CNT-containing fruits or the corresponding residual level of pure CNTs (0.001 µg ml-1) was not sufficient to initiate a significant change in transepithelial resistance and on gene expression of the model T-84 human intestinal epithelial cells. However, at 10 µg ml-1 concentration CNTs were able to penetrate the cell membrane and change the gene expression profile of exposed cells. Moreover, extracts from CNT-containing fruits had minimal to no effect on human intestinal microbiota as revealed by culture-based analysis and 16S rRNA sequencing.

Effects of carbon-based nanomaterials on seed germination, biomass accumulation and salt stress response of bioenergy crops.

Bioenergy crops are an attractive option for use in energy production. A good plant candidate for bioenergy applications should produce a high amount of biomass and resist harsh environmental conditions. Carbon-based nanomaterials (CBNs) have been described as promising seed germination and plant growth regulators. In this paper, we tested the impact of two CBNs: graphene and multi-walled carbon nanotubes (CNTs) on germination and biomass production of two major bioenergy crops (sorghum and switchgrass). The application of graphene and CNTs increased the germination rate of switchgrass seeds and led to an early germination of sorghum seeds. The exposure of switchgrass to graphene (200 mg/l) resulted in a 28% increase of total biomass produced compared to untreated plants. We tested the impact of CBNs on bioenergy crops under salt stress conditions and discovered that CBNs can significantly reduce symptoms of salt stress imposed by the addition of NaCl into the growth medium. Using an ion selective electrode, we demonstrated that the concentration of Na+ ions in NaCl solution can be significantly decreased by the addition of CNTs to the salt solution. Our data confirmed the potential of CBNs as plant growth regulators for non-food crops and demonstrated the role of CBNs in the protection of plants against salt stress by desalination of saline growth medium.

Assessment of Effects of the Long-Term Exposure of Agricultural Crops to Carbon Nanotubes.

Carbon-based nanoparticles (CBNs) are nanomaterials that have been shown to be plant growth regulators. Here, we investigated the effects of long-term exposure to multi-walled carbon nanotubes (MWCNTs) on the growth of three important crops (barley, soybean, and corn). The tested species were cultivated in hydroponics supplemented with 50 µg/mL MWCNTs. After 20 weeks of continuous exposure to the nanomaterials, no significant toxic effects on plant development were observed. Several positive phenotypical changes were recorded, in addition to the enhancement of photosynthesis in MWCNT-exposed crops. Raman spectroscopy with point-by-point mapping proved that the MWCNTs in the hydroponic solution moved into all tested species and were distributed in analyzed organs (leaves, stems, roots, and seeds). Our results confirmed the significant potential of CBN in plant agriculture. However, the documented presence of MWCNTs in different organs of all exposed crops highlighted the importance of detailed risk assessment of nanocontaminated plants moving into the food chain.

Multiwalled Carbon Nanotubes Dramatically Affect the Fruit Metabolome of Exposed Tomato Plants.

Here, we reported that multiwalled carbon nanotubes (MWCNT) added to hydroponics system can enhance fruit production of exposed tomato plants. We quantified the exact amount of MWCNT accumulated inside of fruits collected by MWCNT-exposed plants using an advanced microwave induced heating technique (MIH). We found that absorption of MWCNT by tomato fruits significantly affected total fruit metabolome as was confirmed by LC-MS. Our data highlight the importance of comprehensive toxicological risk assessment of plants contaminated with carbon nanomaterials.

Graphene and carbon nanotubes activate different cell surface receptors on macrophages before and after deactivation of endotoxins.

Nanomaterial synthesis and handling in a non-sterile environment can result in the final product becoming contaminated with bacterial endotoxin or lipopolysaccharides (LPB). During toxicological testing, the effects caused by endotoxin-contaminated nanomaterials can be misinterpreted in the end-point analysis (such as cytotoxicity and immune responses) and could result in erroneous conclusions. The objective of this study was twofold: (i) to test different carbon-based nanomaterials (CBNs) [pristine graphene and multi-wall carbon nanotubes (MWCNTs)] for the presence of endotoxin and develop strategies for depyrogenation, and (ii) to compare the immune response exhibited by macrophages after exposure to native CBNs versus depyrogenated CBNs. The gel-clot limulus amebocyte lysate (LAL) and chromogenic-based LAL assays were used to detect endotoxins. Results revealed that the CBNs contained greater amounts of endotoxin than are approved by major regulatory agencies (0.5 EU ml-1 ). Three repeated cycles of autoclaving reduced the endotoxin in the test materials. Macrophages were incubated with pyrogenated and depyrogenated pristine graphene and MWCNTs to test differences in phagocytosis, cytotoxicity, and expression of genes involved in macrophage activation. The uptake of depyrogenated CBNs was significantly reduced as compared with pyrogenated CBNs. Exposure of macrophages to depyrogenated CBNs resulted in a distinct pattern of gene expression for TLR signaling, NOD-like receptor signaling, and downstream signal transduction molecules. Furthermore, macrophages exposed to both types of CBNs showed the downregulation of TLR5 and NLRC4 inflammasomes. The results of this study reaffirm that assessment of endotoxin and other bacterial contamination is critical when evaluating the cellular toxicity of nanomaterials. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Carbon nanotubes as carriers of Panax ginseng metabolites and enhancers of ginsenosides Rb1 and Rg1 anti-cancer activity.

A major benefit to nanomaterial based-medicine is the ability to provide nanosized vehicles for sporadic metabolites. Here, we describe how the conjugation of valuable ginseng secondary metabolites (ginsenoside Rb1 or Rg1) with carbon nanotubes (CNT) can enhance their anti-proliferative and anti-cancer effects. Ginsenoside-CNT conjugate (Rb-CNT or Rg-CNT) permitted the ginsenosides to be used at a low dose, yet achieve a higher incidence of cancer killing. We were able to demonstrate that the ginsenoside-CNT conjugate can decrease cell viability up to 62% in breast cancer cells (MCF-7) and enhance antiproliferation of drug-resistant pancreatic cancer cells (PANC-1) by 61%. The interaction of the ginsenoside-CNT conjugate with breast cancer cells was studied using Raman Spectroscopy mapping. Total transcriptome profiling (Affymetrix platform) of MCF-7 cells treated with the ginsenoside-CNT conjugate shows that a number of cellular, apoptotic and response to stimulus processes were affected. Therefore, our data confirmed the potential use of CNT as a drug delivery system.

Comparative study of plant responses to carbon-based nanomaterials with different morphologies.

The relationship between the morphology of carbon-based nanomaterials (CBNs) and the specific response of plants exposed to CBNs has not been studied systematically. Here, we prove that CBNs with different morphologies can activate cell growth, germination, and plant growth. A tobacco cell culture growth was found to increase by 22%-46% when CBNs such as helical multi-wall carbon nanotubes (MWCNTs), few-layered graphene, long MWCNTs, and short MWCNTs were added to the growth medium at a concentration of 50 µg ml(-1). The germination of exposed tomato seeds, as well as the growth of exposed tomato seedlings, were significantly enhanced by the addition of all tested CBNs. The presence of CBNs inside exposed seeds was confirmed by transmission electron microscopy and Raman spectroscopy. The effects of helical MWCNTs on gene expression in tomato seeds and seedlings were investigated by microarray technology and real time-PCR. Helical MWCNTs affected a number of genes involved in cellular and metabolic processes and response to stress factors. It was shown that the expression of the tomato water channel gene in tomato seeds exposed to helical MWCNTs was upregulated. These established findings demonstrate that CBNs with different morphologies can cause the same biological effects and share similar mechanisms in planta.

Genetic reduction of inositol triphosphate (InsP3) increases tolerance of tomato plants to oxidative stress.

MAIN CONCLUSION: We demonstrate here that the reduction of InsP 3 , the key component of the phosphoinositol pathway, results in changes in ROS-scavenging machinery and, subsequently, increases the tolerance of tomato plants to light stress. Different plant stress signaling pathways share similar elements and, therefore, 'cross-talk' between the various pathways can exist. Links between the phosphoinositol signaling pathway and light signaling were recently found. Tomato plants expressing InsP 5-ptase and exhibiting reduction in the level of inositol 1,4,5-triphosphate (InsP3) demonstrated enhanced tolerance to stress caused by continuous light exposure. To understand the molecular basis of observed stress tolerance in tomato lines with decreased amount of InsP3, we monitored the expression of enzymatic antioxidants as well as important factors in light signaling associated with non-enzymatic antioxidants (secondary metabolites). Here, we demonstrated that InsP 5-ptase transgenic plants accumulate less hydroxide peroxide and maintain higher chlorophyll content during stress caused by continuous light exposure. This observation can be explained by documented activation of multiple enzymatic antioxidants (LeAPX1, SICAT2, LeSOD) at levels of gene expression and enzymatic activities during continuous light exposure. In addition, we noticed the up-regulation of photoreceptors LePHYB and LeCHS1, key enzymes in flavonoid biosynthesis pathway, transcription factors LeHY5, SIMYB12, and early light-inducible protein (LeELIP) genes in transgenic tomato seedlings exposed to blue or red light. Our study confirmed the existence of a correlation between phosphoinositol signaling pathway modification, increased tolerance to stress caused by continuous light exposure, activation of ROS-scavenging enzymes, and up-regulation of molecular activators of non-enzymatic antioxidants in InsP 5-ptase expressing tomato lines.

Impact of carbon nanotube exposure to seeds of valuable crops.

Multiwalled carbon nanotubes (MWCNTs) affected seed germination, growth, and the development of three important crops (barley, soybean, corn). Early seed germination and activation of growth in exposed seedlings was observed when MWCNTs were added to sterile agar medium. Similarly, seed germination was activated for all tested crop species when MWCNTs were deposited on seed surfaces. The ability of MWCNTs to penetrate the seed coats of corn, barley, and soybean was proven by detection of nanotube agglomerates inside MWCNT-exposed seeds using Raman spectroscopy and transmission electron microscopy (TEM). Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the expression of genes encoding several types of water channel proteins was increased in soybean, corn, and barley seeds coated with MWCNTs compared with uncoated control seeds. Our results indicate that the positive effect of MWCNTs on the germination and growth of seedlings is reproducible between crop species and can be observed for different methods of delivering carbon nanotubes. Such studies could prove the significant potential of carbon nanotubes as regulators of germination and plant growth.