Mitsui-7, heat-treated, and nitrogen-doped multi-walled carbon nanotubes elicit genotoxicity in human lung epithelial cells

Background: The unique physicochemical properties of multi-walled carbon nanotubes (MWCNT) have led to many industrial applications. Due to their low density and small size, MWCNT are easily aerosolized in the workplace making respiratory exposures likely in workers. The International Agency for Research on Cancer designated the pristine Mitsui-7 MWCNT (MWCNT-7) as a Group 2B carcinogen, but there was insufficient data to classify all other MWCNT. Previously, MWCNT exposed to high temperature (MWCNT-HT) or synthesized with nitrogen (MWCNT-ND) have been found to elicit attenuated toxicity; however, their genotoxic and carcinogenic potential are not known. Our aim was to measure the genotoxicity of MWCNT-7 compared to these two physicochemically-altered MWCNTs in human lung epithelial cells (BEAS-2B & SAEC). Results: Dose-dependent partitioning of individual nanotubes in the cell nuclei was observed for each MWCNT material and was greatest for MWCNT-7. Exposure to each MWCNT led to significantly increased mitotic aberrations with multi- and monopolar spindle morphologies and fragmented centrosomes. Quantitative analysis of the spindle pole demonstrated significantly increased centrosome fragmentation from 0.024–2.4 μg/mL of each MWCNT. Significant aneuploidy was measured in a dose-response from each MWCNT-7, HT, and ND; the highest dose of 24 μg/mL produced 67, 61, and 55%, respectively. Chromosome analysis demonstrated significantly increased centromere fragmentation and translocations from each MWCNT at each dose. Following 24 h of exposure to MWCNT-7, ND and/or HT in BEAS-2B a significant arrest in the G1/S phase in the cell cycle occurred, whereas the MWCNT-ND also induced a G2 arrest. Primary SAEC exposed for 24 h to each MWCNT elicited a significantly greater arrest in the G1 and G2 phases. However, SAEC arrested in the G1/S phase after 72 h of exposure. Lastly, a significant increase in clonal growth was observed one month after exposure to 0.024 μg/mL MWCNT-HT & ND. Conclusions: Although MWCNT-HT & ND cause a lower incidence of genotoxicity, all three MWCNTs cause the same type of mitotic and chromosomal disruptions. Chromosomal fragmentation and translocations have not been observed with other nanomaterials. Because in vitro genotoxicity is correlated with in vivo genotoxic response, these studies in primary human lung cells may predict the genotoxic potency in exposed human populations

Thyroid Hormone T3 Counteracts STZ Induced Diabetes in Mouse

This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrified. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes

Gli effetti patrimoniali della separazione: lo scioglimento della comunione legale ai sensi del nuovo art. 191, comma 2, cod.civ.

Nel saggio viene analizzata criticamente la disciplina relativa allo scioglimento della comunione legale a seguito della separazione dei coniugi prima e dopo la riforma introdotta con la l. 6 maggio 2015, n. 5

Apoptosis and survival.

<p><b>A</b> Apoptosis measurement Tissue sections from the different experimental groups of animals have been obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. Caspases activity (left panel, red) was detected by CaspGLOW and Tunel assay (right panel) was visualized by Immunohistochemistry. At 48 h of STZ alone apoptotic nuclei were clearly detectable within the islets, while in the samples exposed contemporary to T3 apoptotic nuclei were hardly detectable within the islets. At least 10 different islets per sample were analyzed for each experiment. Data are from 1 or 2 experiments with similar results (n = 5 animals/group). Space bar: 100 µm. Histogram: The percentage of Tunel or Caspase positive cells was calculated by counting up to a minimum of 200 cells for ten optical fields (200×) for each sample, randomly taken from two different experiments. The effect of treatment with T3 was statistically significant versus STZ. Student's <i>t</i> test: p<0.01 vs STZ. <b>B</b> Western Blot: Western Blot analyses were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> on protein extracts from the various experimental groups and a specific band corresponding to Bax and Casapase3 was detected. As shown, while the presence of STZ clearly induced the expression of Bax and the activation of Casp 3, the presence of T3 was able to counteract STZ action, maintaining Bax and Casp 3 levels comparable to the CTR samples. The expression of B-actin was analyzed as a control for gel loading. At least three different experiments were performed, and a representative one is shown here. Densitometric absorbance values from three separate experiments were averaged (± SD), after they had been normalized to B-actin for equal loading. Data relative to each protein are presented on the right of the Western Blot panel in the histogram as Relative Densitometric Units (y axis). The different experimental groups are indicated on the x axis. A comparison of the individual treatment was conducted by using Student's <i>t</i> test. p = 0.003.</p

Ultrastructure of β cells.

<p>Trasmission electron micrographs of STZ and STZ+T3 treated pancreatic islets compared to control (Uranyl acetate/lead citrate; space bar 1 µm). Nu, nucleus; MB, myelinic bodies. The ultrastructure of the β cells was affected by STZ and maintained unaltered by the addition of T3. Data are from 1 or 2 experiments with similar results (n = 5 animals/group).</p

Akt activation.

<p>Immunohistochemistry: Tissue section from the different experimental groups of animals have been obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. Immunohistochemistry for pAKT (Ser 473) was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a>. The presence of T3 clearly provoked an increment in Akt activation, as compared to total Akt expression (data not shown) Data are from 1 or 2 experiments with similar results (n = 5 animals/group). At least ten fields <i>per</i> chamber and three independent cultures were examined Space bar: 100 µm. Western Blot: Western Blot analyses were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> and a specific band corresponding to the phosphorylated Akt (Ser 473) was detected. The expression of total Akt was analyzed as a control for gel loading. The presence of T3 clearly provoked an increment in Akt activation (Ser 473), as compared to total Akt expression. At least three different experiments were performed, and a representative one is shown here. Densitometric absorbance values from three separate experiments were averaged (± SD), after they had been normalized to Akt for equal loading. Data relative to each protein are presented on the right of the Western Blot panel in the histogram as Relative Densitometric Units (y axis). The different experimental groups are indicated on the x axis. A comparison of the individual treatment was conducted by using Student's <i>t</i> test. P<0.001.</p

Phisiological parameters.

<p><b>A, B</b>: Analysis of blood glucose and Insulin levels after intra-peritoneal glucose tolerance test (upper panels). Glycemia was measured by glucometer, while Insulin concentration was assessed by ELISA assay, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. Oral administration of T3 significantly reduces severity and progression of STZ-induced diabetes in Balb/c mice and assured normal Insulin responsiveness. <b>C</b>: Insulin tolerance was performed (lower panel) after intra peritoneal glucose injection. Insulin was injected intraperitoneally after glucose to the different experimental groups of animals. Glycemia was measured by glucometer. Results represent the mean ± SE of three separate experiments. Grey: control black:STZ white:STZ+T3. <b>D</b> RT-PCR: Total RNA was extracted from liver from mice of the different experimental groups and RTPCR was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. A single product was obtained for each gene, as showed by agarose electrophoresis. All PCR products were of the expected size and sequence. The presence of T3 did not induce any change in the DIO1 expression, as normalized to 18s.</p

Islets analyses.

<p><b>A</b> Histopathology: Tissue section from the different experimental groups of animals have been obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. Immunofluorescence for Insulin (green, left panels), and Glucagon (green, middle panel), and Hematoxilin and Eosin staining (right panels) were performed to analyze histopathological changes in pancreatic islets compared to control mice (CTR). Nuclei were counterstained with Hoechst (blue) in the IF experiments. Data are from 1 or 2 experiments with similar results (n = 5 animals/group). At least ten fields <i>per</i> chamber and three independent cultures were examined Space bar: 100 µm. Histogram: The percentage of Insulin positive cells was calculated by counting up to a minimum of 200 cells for ten optical fields (200×) for each sample, randomly taken from two different experiments. The effect of treatment with T3 was statistically significant versus STZ. Student's <i>t</i> test: p<0.05 vs STZ+T3. <b>B</b> Islet crossection area: Crossection area was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> and the results were averaged and represented on the histogram. The presence of T3 significantly counteracts the reduction of islet area and deterioration. At least 10 different islets per sample were analyzed for each experiment. Data are from 1 or 2 experiments with similar results (n = 5 animals/group). The effect of treatment with T3 was statistically significant versus STZ+T3. Student's <i>t</i> test: p<0.01 vs STZ+T3. <b>C</b> Real Time PCR: Total RNA was obtained from pancreata from animals of the various experimental groups and RT-qPCR was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. Melting point analysis of PCR products for both genes demonstrated single product formation, as confirmed by gel electrophoresis (on the right). All PCR products were of the expected size and sequence. Normalized ratios are shown in the histogram; the presence of T3 was able to overcome the STZ inhibition of Insulin gene expression.</p

Glut-2 status.

<p>Immunofluorescence: Tissue sections from the different experimental groups of animals have been obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. Indirect Immunofluorescence for Glut-2 (green) revealed the presence of the Glucose transporter within the islets, clearly detectable. Nuclei were counterstained with Hoechst (blue). As shown with T3 treatment, no differences either in the expression levels or in the localization of the Glucose transporter Glut-2 was observed. On the other hand, the presence of the STZ was sufficient to cause a strong disarray in both expression and localization of Glut-2 in the islets. Data are from 1 or 2 experiments with similar results (n = 5 animals/group). At least ten fields <i>per</i> chamber and three independent cultures were examined. Space bar: 100 µm. Western Blot: Western Blot analyses were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> on protein extracts from the CTR and the T3 treated animals and a specific band corresponding to Glut-2 was detected. The presence of T3 did not provoke any change in the Glut-2 expression, as compared to B-actin expression. The expression of B-actin was analyzed as a control for gel loading. At least three different experiments were performed, and a representative one is shown here. Densitometric absorbance values from three separate experiments were averaged (± SD), after they had been normalized to B-actin for equal loading. Data are presented on the right of the Western Blot panel in the histogram as Relative Densitometric Units (y axis). The different experimental groups are indicated on the x axis. A comparison of the individual treatment was conducted by using Student's <i>t</i> test. P<0.003.</p