Santosh Phuyal

• Alswady-Hoff, Mayes; Erdem, Johanna Maria Samulin; Phuyal, Santosh; Knittelfelder, Oskar; Sharma, Animesh; Fonseca, Davi de Miranda; Skare, Øivind; Slupphaug, Geir & Zienolddiny, Shanbeh (2021). Long-Term Exposure to Nanosized TiO2 Triggers Stress Responses and Cell Death Pathways in Pulmonary Epithelial Cells. International Journal of Molecular Sciences.  ISSN 1422-0067.  22(10) . doi: 10.3390/ijms22105349 Show summary

  There is little in vitro data available on long-term effects of TiO2 exposure. Such data are important for improving the understanding of underlying mechanisms of adverse health effects of TiO2 . Here, we exposed pulmonary epithelial cells to two doses (0.96 and 1.92 µg/cm2 ) of TiO2 for 13 weeks and effects on cell cycle and cell death mechanisms, i.e., apoptosis and autophagy were determined after 4, 8 and 13 weeks of exposure. Changes in telomere length, cellular protein levels and lipid classes were also analyzed at 13 weeks of exposure. We observed that the TiO2 exposure increased the fraction of cells in G1-phase and reduced the fraction of cells in G2-phase, which was accompanied by an increase in the fraction of late apoptotic/necrotic cells. This corresponded with an induced expression of key apoptotic proteins i.e., BAD and BAX, and an accumulation of several lipid classes involved in cellular stress and apoptosis. These findings were further supported by quantitative proteome profiling data showing an increase in proteins involved in cell stress and genomic maintenance pathways following TiO2 exposure. Altogether, we suggest that cell stress response and cell death pathways may be important molecular events in long-term health effects of TiO2

• Eck, Franziska; Phuyal, Santosh; Smith, Matthew D.; Kaulich, Manuel; Wilkinson, Simon; Farhan, Hesso & Behrends, Christian (2020). ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis. Journal of Cell Science.  ISSN 0021-9533.  133(18), s 1- 16 . doi: 10.1242/jcs.243477 Full text in Research Archive.
• Phuyal, Santosh & Baschieri, Francesco (2020). Endomembranes: Unsung Heroes of Mechanobiology?. Frontiers in Bioengineering and Biotechnology.  ISSN 2296-4185.  8 . doi: 10.3389/fbioe.2020.597721 Full text in Research Archive.
• Phuyal, Santosh & Farhan, Hesso (2019). Multifaceted Rho GTPase Signaling at the Endomembranes. Frontiers in Cell and Developmental Biology.  ISSN 2296-634X.  7(127) . doi: 10.3389/fcell.2019.00127 Full text in Research Archive.
• Phuyal, Santosh; Kasem, Mayes; Knittelfelder, Oskar; Sharma, Animesh; Fonseca, Davi de Miranda; Vebraite, Vaineta; Shaposhnikov, Sergey; Slupphaug, Geir; Skaug, Vidar & Zienolddiny, Shanbeh (2018). Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes. Nanotoxicology.  ISSN 1743-5390.  12(2), s 138- 152 . doi: 10.1080/17435390.2018.1425500 Show summary

  The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.

• Moen, Aurora Lie; Jacobsen, Daniel; Phuyal, Santosh; Legfeldt, Anna; Haugen, Fred; Røe, Cecilie & Gjerstad, Johannes (2017). MicroRNA-223 demonstrated experimentally in exosome-like vesicles is associated with decreased risk of persistent pain after lumbar disc herniation. Journal of Translational Medicine.  ISSN 1479-5876.  15(89), s 1- 11 . doi: 10.1186/s12967-017-1194-8 Full text in Research Archive. Show summary

  Background: Previous findings have demonstrated that lumbar radicular pain after disc herniation may be associated with up-regulation of inflammatory mediators. In the present study we examined the possible role of extracellular microRNAs (miRs) in this process. Methods: Single unit recordings, isolation of exosome-like vesicles, electron microscopy, nanoparticle tracking analysis, western blot analysis and qPCR were used in rats to demonstrate the effect of nucleus pulposus (NP) applied onto the dorsal nerve roots. ELISA and qPCR were used to measure the level of circulating IL-6 and miRs in a 1-year observational study in patients after disc herniation. Results: In the rats, enhanced spinal cord nociceptive responses were displayed after NP applied onto the dorsal nerve roots. An increased release of small non-coding RNAs, including miR-223, miR-760 and miR-145, from NP in exosome-like vesicles was demonstrated. In particular, the NP expression of miR-223, which inhibited the nociceptive spinal signalling, was increased. In the patients, increased extracellular miR-223 was also verified in the acute phase after disc herniation. The increased miR-223 expression was, however, only observed in those who recovered (sex, age and smoking were included as covariates). Conclusions: Our findings suggest that miR-223, which can be released from the NP after disc herniation, attenuates the neuronal activity in the pain pathways. Dysregulation of miR-223 may predict chronic lumbar radicular pain.

• Phuyal, Santosh; Kasem, Mayes; Rubio, Laura; Karlsson, Hanna; Marcos, Ricard; Skaug, Vidar & Zienolddiny, Shanbeh (2017). Effects on human bronchial epithelial cells following low-dose chronic exposure to nanomaterials: A 6-month transformation study. Toxicology in Vitro.  ISSN 0887-2333.  44, s 230- 240 . doi: 10.1016/j.tiv.2017.07.016 Show summary

  The most plausible exposure route to manufactured nanomaterials (MNM) remains pulmonary inhalation. Yet, few studies have attempted to assess carcinogenic properties in vitro following long-term exposure of human pulmonary cells to low and occupationally relevant doses. The most advanced in vitro tests for carcinogenicity, the cell transformation assay (CTA), rely mostly on rodent cells and short-term exposure. We hypothesized that long-term exposure of human bronchial epithelial cells with a normal phenotype could be a valuable assay for testing carcinogenicity of nanomaterials. Therefore, this study (performed within the framework of the FP7-NANoREG project) assessed carcinogenic potential of chronic exposure (up to 6 months) to low doses of multi-walled carbon nanotubes (MWCNT, NM-400 and NM-401) and TiO2 materials (NM62002 and KC7000). In order to harmonize and standardize the experiments, standard operating protocols of MNM dispersion (NANOGENOTOX) were used by three different NANoREG project partners. All nanomaterials showed low cytotoxicity in short-term tests for the tested doses (0.96 and 1.92 μg/cm2). During long-term exposure, however, NM-401 clearly affected cell proliferation. In contrast, no cell transformation was observed for NM-401 by any of the partners. NM-400 and NM62002 formed some colonies after 3 months. We conclude that agglomerated NM-401 in low doses affect cell proliferation but do not cause cell transformation in the CTA assay used.

• Vestad, Beate; Llorente, Alicia; Neurauter, Axl; Phuyal, Santosh; Kierulf, Bente; Kierulf, Peter; Skotland, Tore; Sandvig, Kirsten; Foss, Kari Bente & Øvstebø, Reidun (2017). Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. Journal of Extracellular Vesicles.  ISSN 2001-3078.  6(1), s 1- 11 . doi: 10.1080/20013078.2017.1344087 Full text in Research Archive.
• Phuyal, Santosh; Skotland, Tore; Hessvik, Nina Pettersen; Simolin, Helena; Øverbye, Anders; Brech, Andreas; Parton, Robert G.; Ekroos, Kim; Sandvig, Kirsten & Llorente, Alicia (2015). The ether lipid precursor hexadecylglycerol stimulates the release and changes the composition of exosomes derived from PC-3 cells. Journal of Biological Chemistry.  ISSN 0021-9258.  290, s 4225- 4237 . doi: 10.1074/jbc.M114.593962
• Phuyal, Santosh; Hessvik, Nina Pettersen; Skotland, Tore; Sandvig, Kirsten & Llorente, Alicia (2014). Regulation of exosome release by glycosphingolipids and flotillins. The FEBS Journal.  ISSN 1742-464X.  281(9), s 2214- 2227 . doi: 10.1111/febs.12775
• Hessvik, Nina Pettersen; Phuyal, Santosh; Brech, Andreas; Sandvig, Kirsten & Llorente, Alicia (2012). Profiling of microRNAs in exosomes released from PC-3 prostate cancer cells. Biochimica et Biophysica Acta. Gene Regulatory Mechanisms.  ISSN 1874-9399.  1819(11-12), s 1154- 1163 . doi: 10.1016/j.bbagrm.2012.08.016

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• Zienolddiny, Shanbeh; Phuyal, Santosh; Kasem, Mayes; Knittelfelder, Oskar; Sharma, Animesh; Fonseca, Davi de Miranda; Vebraite, Vaineta & Shaposhnikov, Sergey (2017). Toxicoproteomic effects of carbon nanotubes on chronically exposed human lung cells. Toxicology Letters.  ISSN 0378-4274.  280, s S141 . doi: 10.1016/j.toxlet.2017.07.395
• Phuyal, Santosh; Llorente, Alicia & Sandvig, Kirsten (2014). Composition and cellular release of exosomes from cancer cells.

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