Toxic and genotoxic effects of graphene and multi-walled carbon nanotubes

<p>Graphene and multi-walled carbon nanotubes (MWCNT) are widely used in nanomedicine, and other fields, due to their unique physicochemical properties including high tensile strength, ultra-light weight, thermal and chemical stability, and reliable semi-conductive electronic properties. Although extensive amount of data exist describing their adverse effects including potential genotoxicity, few studies using gene mutation detection approaches in mammalian cells are available, which represents an important gap for risk estimations. The aim of the present study was to determine the effects of graphene or MWCNT [as pure, carboxyl (COOH) functionalized, and amide (NH₂) functionalized] on cytotoxicity, intracellular levels of reactive oxygen species, apoptosis, gene expression changes, and gene mutation induction in L5178Y/<i>Tk^+/–</i>3.7.2C mouse lymphoma cell line. Although some adverse effects were observed at concentrations of 350 and 450 µg/ml, which are excessive and not environmentally relevant levels, no marked effects were detected at concentrations of 250 µg/ml and lower. This is the first study reporting cytotoxicity, mutagenicity, and gene expression findings in the mouse lymphoma cell line for graphene and different MWCNT forms at high concentrations; however, the biological relevance of these observations needs to be assessed following chronic <i>in vivo</i> exposure.</p

Antigenotoxic potential of boron nitride nanotubes

<p>Boron and boron nitride nanotubes (BNNTs) are increasingly used in different industrial fields and, potentially, in some biomedical areas. As occurs with other nanomaterials (NMs), to increase our knowledge on their potential health hazards is a priority. Although <i>in vitro</i> approaches are a routine in getting biological information on the biological effects of NMs, the use of simple <i>in vivo</i> model organisms is receiving an increased interest. In this context, <i>Drosophila melanogaster</i> is widely used as a eukaryotic model for the study of the potential harmful effects associated with various agents, including NMs. The aim of this study is to provide new data on the potential antioxidant/antigenotoxic properties of boron and boron nitride nanotubes (BNNTs), as well as on other biological end-points. Our results show changes in the expression of genes involved in the antioxidant defense (<i>CAT</i> and <i>SOD</i>), and in those rel0061ted to the integrity of the intestinal barrier (<i>Duox</i>, <i>Hml</i>, <i>Muc68D</i>, and <i>PPO2</i>), at the highest exposure doses (5, 10 mM). However, non-relevant toxic or genotoxic effects were observed. Interestingly, BNNTs and boron significantly reduced the genotoxic effect of potassium dichromate (PDC), and the intracellular levels of reactive oxygen species (ROS). This suggest that the observed effects can be linked to the antioxidant properties of BNNTs and boron. This is the first study reporting antigenotoxicity/genotoxicity, and gene expression data, in the somatic cells of <i>D. melanogaster</i> larvae for BNNTs.</p

Telomere length distribution in the newborn population (N = 74).

<p>As observed, TRF length is heterogeneous from birth.</p

Correlation between TRF length and newborn baseline genetic damage.

<p>A negative significant association is observed, were individuals with shorter telomeres present significantly higher levels of DNA damage. The mean ± SEM values of telomere length and basal DNA damage are also indicated for the overall population (N = 74) and for the individuals with shorter (N = 18) and longer (N = 19) telomeres. Individuals with shorter telomeres show significant higher level of DNA damage. Student <i>t</i>-test for long <i>vs</i> short telomere groups; *<i>P</i><0.05, **<i>P</i>< 0.01.</p

Correlation between TRF length and newborn induced genetic damage.

<p>A subgroup of 35 cord blood samples were selected and challenged to MMC treatment to establish their sensitivity to induced genomic instability. As observed, individuals with shorter telomeres present higher levels of MMC-induced genetic damage. The mean ± SEM values of telomere length, basal and induced DNA damage are also indicated for the overall population (N = 35) and for the individuals with shorter (N = 10) and longer (N = 10) telomeres. Individuals with shorter telomeres show significant higher level of basal and induced DNA damage. Student <i>t</i>-test for long vs. short telomere groups; **<i>P</i><0.01, ***<i>P</i><0.001.</p

Additional file 1: of Effects of differently shaped TiO2NPs (nanospheres, nanorods and nanowires) on the in vitro model (Caco-2/HT29) of the intestinal barrier

Table S1. Primer sequences. Table S2. Interconversion of the used concentrations. The relationships between μg/mL and μg/cm2 are indicated. Figure S1. Monolayer confirmation of the intestinal in vitro model, Caco-2/HT29. Transversal cuts of the Caco-2/HT29 barrier stained in Alcian Blue (A and C). Transversal cuts of Caco-2 monocultures (B and D) stained in Hematoxylin and Eosin. Figure S2. Dynamic light scattering characterization of the NP’s over the incubation time. Hydrodynamic size for TiO2NPs-S (A), TiO2NPs-R (B), and TiO2NPs-W (C), suspended in DMEM cell culture medium at concentrations ranging from 12.5 to 350 μg/mL. Bars that do not share any letter are significantly different according to the one-way ANOVA with a Tukey’s post-test (P < 0.05). Data is represented as mean ± SD. Figure S3. Three-dimensional confocal image of the Caco-2/HT29 co-culture exposed to 150 μg/mL of TiO2NPs-Wires. Cell nuclei (blue) were stained with Hoechst and mucus (red) stained with WGA. NPs were visualized by reflection and marked with a green mask. NPs-cell nucleus interactions are indicated with white circles. Images were processed with the Imaris 7.2.1 software. Figure S4. Confocal images of the reflected NPs found in the collected basolateral medium after exposing the Caco-2/HT29 co-culture barrier to 150 μg/mL of TiO2NPs. (DOCX 3832 kb

Chromatin inmunoprecipitation (ChIP) assays.

<p>(A) The PCR amplified regions comprising the predicted NFκB binding sites (kBS1, kBS2 and kBS3) are indicated as rectangles, with location of these specific DNA segments indicated by numbers. (B) TPC and CGTH cells were treated with TNF-α and ChIP assays performed using IgG (mock) and NFκBp65 antibodies. Imnmunoprecipitated DNA was analyzed by qPCR with primers targeted to the predicted NFκB binding sites (kBS1, kBS2 and kBS3). The quantitative data reflect occupancy calculated as % input and represented as mean±SD of two independent experiments. *: P<0.05; **: P<0.01.</p

Identification of functional NF-κB responsive elements in the 5’-flanking region of the human <i>TBX15</i> gene.

<p>(A) Location and sequence of the predicted NF-κB binding sites (kBS1, kBS2 and kBS3) in the studied region of the <i>TBX15</i> promoter, with indication of altered sequence in mutated reporter constructs (underlined). Site directed mutagenesis was performed using P2 reporter construct to obtain the mutated reporter constructs. (B) Luciferase analysis of each mutated reporter constructs. <i>Left panel</i>, co-tansfected with the pCDNA3-p65 expression vector (p65) or the pCDNA3 empty vector (EV). <i>Right panel</i>, in nonstimulated or stimulated TNF-α cells. At least three independent experiments were performed with duplicates. Bars represent the mean±SD. * indicates p-value < 0.05 and ** p-value < 0.01.</p

Expression of <i>TBX15</i>mRNA and <i>CXCL1</i>mRNA in BHT cell lines after stimulation with TNF-α and PMA/I.

<p>(A) qRT-PCR of <i>TBX15</i>mRNA analysis in BHT and BHT IkBαSR cells after 3h of TNF-α or PMA/I treatment. (B) qRT-PCR of <i>CXCL1</i>mRNA analysis in BHT and BHT IkBαSR cells after 3h of TNF-α or PMA/I treatment. Data were normalized to <i>RPL27</i> and expressed as fold change referred to untreated cells whose <i>TBX15</i>mRNA or <i>CXCL1</i>mRNA relative expression was defined as 1. Data are mean ± SD of mRNA levels in triplicates. ** indicates p-value < 0.01.</p

Expression of <i>TBX15</i>mRNA in different cell lines after stimulation with TNF-α.

<p>(A) qRT-PCR of <i>TBX15</i>mRNA analysis in FRO, CGTH, TPC-1 and HeLa cells after 2h of TNF-α treatment. (B) qRT-PCR of <i>TBX15</i>mRNA (left) and <i>CXCL1</i>mRNA (right) analysis in p65^+/+ and p65^<i>-/-</i> MEF cells after TNF-α treatment. Data were normalized to <i>RPL27</i> and expressed as fold change referred to untreated cells whose <i>TBX15</i>mRNA or <i>CXCL1</i>mRNA relative expression was defined as 1. Data are mean±SD of mRNA levels of two independent experiments in triplicates. ** indicates p-value < 0.01.</p