Proteomic Characterization of Biological Effects Induced by Engineered Nanomaterials

Nanotechnology has been one of the major success stories of the early 21st century. The foundation for this success rests on the discovery that a small size confers completely new properties on materials. Nowadays, engineered nanomaterials (ENMs) are used in a plethora of applications such as paints, cosmetics, food products and electronics. These new properties, however, potentially make ENMs more reactive in biological systems than their large-scale counterparts. Already, asbestos-like effects have been described in mice after exposure to certain forms of carbon nanotubes (CNTs), while nano-sized titanium dioxide (nTiO2) has been shown to evoke inflammation in mouse lung. Therefore, extensive nanosafety studies have to be performed to ensure that no adverse effects are suffered by either workers or end-users of ENM products. This thesis has investigated the biological effects of ENMs by proteomic methods, first by evaluating the uptake and interactions of ENMs with plasma and cellular proteins followed by an analysis of the effects of ENM exposure on the intracellular proteome and secretome of human primary macrophages. The results revealed that ENM interactions with cellular proteins were governed by the surface reactivity of ENMs, whereas interactions with plasma proteins seemed to depend on the combination of both surface reactivity and active recognition, namely tagging of ENMs by opsonin proteins. The binding of cellular proteins to ENMs and subsequent interference with cellular processes might represent a novel cause of ENM toxicity, especially since transmission electron microscopy (TEM) micrographs indicated that several ENM species could be visualized free in the cytoplasm. The cytoplasmic protein expression changes after exposure to silica coated and uncoated nTiO2 revealed that silica coated TiO2 induced stronger protein expression changes in the macrophages. Most of the proteins with altered expression were related to phagocytosis, oxidative stress and inflammation. These proteome changes indicate that macrophages are actively engulfing ENMs and processing them. Moreover, the up-regulation of oxidative stress related proteins might be an indication of oxidative burst. Finally, nTiO2 treatment evoked acetylation of cytoplasmic proteins, a previously uncharacterized phenomenon in cells exposed to ENMs. The results from the macrophage secretome analysis showed that asbestos and long rigid carbon nanotubes (R CNTs) produced a similar response, while protein secretion profile of macrophages exposed to long tangled carbon nanotubes (T CNTs) exhibited a distinct profile. Bioinformatic analysis revealed that R CNTs evoked secretion of inflammation and apoptosis related proteins, possibly because of lysosomal damage. Functional assay confirmed that R CNT exposure triggered apoptosis in macrophages, while T CNTs and asbestos did not. This thesis offers new knowledge concerning the biological effects of engineered nanomaterials. Proteomic methods proved to be useful in the ENM-protein interaction studies revealing that it would be beneficial to include the ENM-protein interaction experiments as part of the routine ENM characterization when assessing the health effects of ENMs. By employing quantitative proteomics, we obtained a global view of both cytoplasmic and secreted proteome changes of macrophages exposed to different ENMs.Nanoteknologia on ollut 2000-luvun alun suurimpia menestystarinoita, joka sai alkunsa kun keksittiin, että pieni koko antaa materiaaleille kokonaan uudenlaisia ominaisuuksia. Nykyään teollisia nanomateriaaleja (TNM) käytetään lukuisissa sovelluksissa, kuten maaleissa, kosmetiikassa, elintarvikkeissa ja elektroniikassa. Pieni koko saattaa kuitenkin aiheuttaa myös ongelmia, sillä nanokokoiset materiaalit saattavat reagoida biologisissa ympäristöissä eri tavalla kuin saman aineen suurempi muoto. Eräiden hiilinanoputkimuotojen onkin jo havaittu aiheuttavan asbestinkaltaisia vaikutuksia hiirimalleissa, lisäksi nanokokoisen titaanidioksidin (nTiO(2)) on raportoitu aiheuttavan keuhkotulehdusta hiirikokeissa. Muun muassa näistä syistä, teollisten nanomateriaalien turvallisuutta olisi tutkittava laajasti jotta vältytään terveysriskeiltä, joille ovat alttiina sekä nanoteollisuudessa työskentelevät että kuluttajat. Tässä väitöskirjassa on tarkasteltu teollisten nanomateriaalien biologisia vaikutuksia proteomiikan menetelmillä. Ensimmäisessä osatyössä tutkittiin TNM:ien pääsyä soluihin ja vuorovaikutuksia proteiinien kanssa. Toisessa osatyössä analysoitiin TNM altistuksen aiheuttamia muutoksia makrofagien soluliman proteomissa ja viimeisessä tarkasteltiin TNM altistuksen aikaansaamia vaikutuksia makrofagien erittämiin proteiineihin. Tulokset osoittivat että tärkein teollisten nanomateriaalien ja soluproteiinien vuorovaikutuksia määrittävä tekijä oli TNM:ien pinnan reaktiivisuus. Sitä vastoin TNM:ien ja plasmaproteiinien vuorovaikutukset perustuivat sekä materiaalien pinnan reaktiivisuuteen että tiettyjen plasmaproteiinien, kuten opsonisaatiossa toimivien proteiinien, kykyyn tunnistaa aktiivisesti vieraita molekyylejä. Teollisten nanomateriaalien tarttuminen solun proteiineihin voi mahdollisesti aiheuttaa toksisia reaktioita häiritsemällä proteiinien toimintaa, varsinkin kun läpäisyelektronimikroskooppikuvissa useat eri nanomateriaalimuodot näyttivät päässeen ulos kalvorakenteiden sisältä solulimaan. Vertailtaessa silikalla päällystetyn nTiO(2):n ja päällystämättömän nTiO(2):n vaikutuksia makrofageissa, voitiin havaita että silikalla päällystetty nTiO(2) aiheutti voimakkaamman muutoksen soluliman proteomissa kuin päällystämätön. Makrofageissa tapahtuvat proteomimuutokset viittasivat aktiiviseen nTiO(2) hiukkasten fagosytoosiin eli solusyöntiin, makrofageissa tapahtuvaan happirasitukseen sekä tulehdusreaktioihin. Happirasituksessa aktivoituvien proteiinien määrän kasvu kertoi mahdollisesti happiradikaalien määrän hetkellisestä räjähdysmäisestä kasvusta (oxidative burst), joka on yleinen makrofageissa tapahtuva reaktio fagosytoosin jälkeen. nTiO(2) altistus aiheutti myös solunsisäisten proteiinien asetyloitumista, jota ei ole aikaisemmin havaittu nanomateriaaleilla altistetuissa soluissa. Kun verrattiin asbestin, pitkien ja jäykkien hiilinanoputkien (R CNT) sekä pitkien ja lankakerämäisten hiilinanoputkien (T CNT) aiheuttamia muutoksia makrofagien erittämien proteiinien määrään ja laatuun, voitiin havaita että R CNT ja asbesti aiheuttivat samankaltaisen reaktion makrofageissa. T CNT:n aiheuttamat muutokset makrofagien erittämissä proteiineissa sen sijaan olivat erilaisia. Bioinformatiikka-analyysit osoittivat että R CNT:lla altistetut makrofagit erittivät tulehdukseen ja solukuolemaan liittyviä proteiineja, mikä mahdollisesti johtui lysosomien hajoamisesta. Lisäkokeilla pystyttiin osoittamaan, että R CNT oli ainoa testatuista materiaaleista joka sai aikaan merkittävää solukuolemaa makrofageissa. Tämä väitöskirja on tuottanut uutta tietoa teollisten nanomateriaalien biologisista vaikutuksista. Käytetyt proteomiikan menetelmät osoittautuivat toimiviksi tutkittaessa TNM:ien ja proteiinien vuorovaikutuksia. Koska teollisten nanomateriaalien pinnan reaktiivisuus vaikuttaa myös materiaalien toksisuuteen, nämä vuorovaikutuskokeet voisivat olla osa koepaneelia, kun selvitetään TNM:ien terveysvaikutuksia. Proteomiikan avulla saatiin myös kokonaisvaltainen näkökulma TNM:ien vaikutuksista makrofagien sisäiseen proteomiin sekä proteiinieritykseen

Elucidating differential nano-bio interactions of multi-walled andsingle-walled carbon nanotubes using subcellular proteomics

Understanding the relationship between adverse exposure events and specific material properties will facilitate predictive classification of carbon nanotubes (CNTs) according to their mechanisms of action, and a safe-by-design approach for the next generation of CNTs. Mass-spectrometry-based proteomics is a reliable tool to uncover the molecular dynamics of hazardous exposures, yet challenges persist with regards to its limited dynamic range when sampling whole organisms, tissues or cell lysates. Here, the simplicity of the sub-cellular proteome was harnessed to unravel distinctive adverse exposure outcomes at the molecular level, between two CNT subtypes. A549, MRC9 and human macrophage cells, were exposed for 24h to non-cytotoxic doses of single-walled or multi-walled CNTs (swCNTs or mwCNTs). Label-free proteomics on enriched cytoplasmic fractions was complemented with analyses of reactive oxygen species (ROS) production and mitochondrial integrity. The extent/number of modulated proteoforms indicated the single-walled variant was more bioactive. Greater enrichment of pathways corresponding to oxido-reductive activity was consistent with greater intracellular ROS induction and mitochondrial dysfunction capacities of swCNTs. Other compromised cellular functions, as revealed by pathway analysis were; ribosome, spliceosome and DNA repair. Highly upregulated proteins (fold change in abundance >6) such as, APOC3, RBP4 and INS are also highlighted as potential markers of hazardous CNT exposure. We conclude that, changes in cytosolic proteome abundance resulting from nano-bio interactions, elucidate adverse response pathways and their distinctive molecular components. Our results indicate that CNT-protein interactions might have a thus far unappreciated significance for protein trafficking, and this warrants further investigation.Peer reviewe

Multi-omics analysis of ten carbon nanomaterials effects highlights cell type specific patterns of molecular regulation and adaptation

New strategies to characterize the effects of engineered nanomaterials (ENMs) based on omics technologies are emerging. However, given the intricate interplay of multiple regulatory layers, the study of a single molecular species in exposed biological systems might not allow the needed granularity to successfully identify the pathways of toxicity (PoT) and, hence, portraying adverse outcome pathways (AOPs). Moreover, the intrinsic diversity of different cell types composing the exposed organs and tissues in living organisms poses a problem when transferring in vivo experimentation into cell-based in vitro systems. To overcome these limitations, we have profiled genome-wide DNA methylation, mRNA and microRNA expression in three human cell lines representative of relevant cell types of the respiratory system, A549, BEAS-2B and THP-1, exposed to a low dose of ten carbon nanomaterials (CNMs) for 48 h. We applied advanced data integration and modelling techniques in order to build comprehensive regulatory and functional maps of the CNM effects in each cell type. We observed that different cell types respond differently to the same CNM exposure even at concentrations exerting similar phenotypic effects. Furthermore, we linked patterns of genomic and epigenomic regulation to intrinsic properties of CNM. Interestingly, DNA methylation and microRNA expression only partially explain the mechanism of action (MOA) of CNMs. Taken together, our results strongly support the implementation of approaches based on multi-omics screenings on multiple tissues/cell types, along with systems biology-based multi-variate data modelling, in order to build more accurate AOPs.Peer reviewe

Integration of genome-wide mRNA and miRNA expression, and DNA methylation data of three cell lines exposed to ten carbon nanomaterials

We present data derived from an exposure experiment in which three cell-lines representative of cell types of the respiratory tissue (epithelial type-I A549, epithelial type-II BEAS-2B, and macrophage THP-1) have been exposed to ten different carbon-based nanomaterials for 48 h. In particular, we provide: genome-wide mRNA and miRNA expression, and DNA methylation; gene tables, containing information on the aberrations induced in these three genomic data layers at the gene level; mechanism of action (MOA) maps representing the comparative functional alteration induced in each cell line and each exposure. (C) 2018 Published by Elsevier Inc.Peer reviewe

Incidence and risk factors of spinal cord stimulation for persistent or recurrent pain after lumbar spine surgery : a population-based study

Purpose This study aims to elucidate the incidence of and independent risk factors for spinal cord stimulator implantations for patients who underwent lumbar spine surgery. Methods The PERFormance, Effectiveness, and Cost of Treatment (PERFECT) episodes database, which was established for selected diseases and procedures in Finland, includes all patients who underwent lumbar spine surgery for degenerative spine conditions or spinal cord stimulation (SCS) in Finland from 1986 to 2018. The data on age, sex, hospital diagnoses, surgical procedures, and causes of death were imported from the Finnish national registers into the PERFECT database. Results Between 1986 and 2018, 157,824 patients had their first lumbar spine procedure and for 1769 (1.1%) of them, a subsequent SCS procedure was observed during the follow-up. The cumulative incidence of SCS for persistent or recurrent pain after lumbar disk herniation, spinal stenosis, degenerative disk disease, and spondylolysis and spondylolisthesis surgery at 15 years was 1.2%, 1.0%, 2.7%, and 2.6% respectively. At 15 years, the cumulative incidence of SCS for persistent or recurrent pain after lumbar spine surgery after five or more lumbar spinal operations was 11.9%. Conclusion Repeated surgery was the most prominent significant risk factor for SCS for persistent or recurrent pain after lumbar spine surgery. The risk of SCS for persistent or recurrent pain after lumbar spine surgery increases significantly along with the number of lumbar spine procedures. When considering repeated lumbar spine surgery, careful evaluation of treatment options should take place to ensure good patient outcomes.Peer reviewe

Soluble triggering receptor expressed on myeloid cells-1 is a marker of organ injuries in cardiogenic shock : results from the CardShock Study

Aims Optimal outcome after cardiogenic shock (CS) depends on a coordinated healing response in which both debris removal and extracellular matrix tissue repair play a crucial role. Excessive inflammation can perpetuate a vicious circle, positioning leucocytes as central protagonists and potential therapeutic targets. High levels of circulating Triggering Receptor Expressed on Myeloid cells-1 (TREM-1), were associated with death in acute myocardial infarction confirming excessive inflammation as determinant of bad outcome. The present study aims to describe the association of soluble TREM-1 with 90-day mortality and with various organ injuries in patients with CS. Methods and results This is a post-hoc study of CardShock, a prospective, multicenter study assessing the clinical presentation and management in patients with CS. At the time of this study, 87 patients had available plasma samples at either baseline, and/or 48 h and/or 96-120 h for soluble TREM-1 (sTREM-1) measurements. Plasma concentration of sTREM-1 was higher in 90-day non-survivors than survivors at baseline [median: 1392 IQR: (724-2128) vs. 621 (525-1233) pg/mL, p = 0.008), 48 h (p = 0.019) and 96-120 h (p = 0.029). The highest tertile of sTREM-1 at baseline (threshold: 1347 pg/mL) was associated with 90-day mortality with an unadjusted HR 3.08 CI 95% (1.48-6.42). sTREM-1 at baseline was not associated to hemodynamic parameters (heart rate, blood pressure, use of vasopressors or inotropes) but rather with organ injury markers: renal (estimated glomerular filtration rate, p = 0.0002), endothelial (bio-adrenomedullin, p = 0.018), myocardial (Suppression of Tumourigenicity 2, p = 0.002) or hepatic (bilirubin, p = 0.008). Conclusion In CS patients TREM-1 pathway is highly activated and gives an early prediction of vital organ injuries and outcome. [GRAPHICS] .Peer reviewe

Hiilinanoputkien aiheuttamien terveysvaikutusten karakterisointi systeemitoksikologian avulla

Hiilinanoputket ovat monikäyttöisiä teollisia nanomateriaaleja, jotka soveltuvat lukuisiin teollisuusaloihin. Hiilinanoputkien asbestin kaltainen muoto on kuitenkin nostanut esiin kysymyksiä niiden turvallisuudesta.1

Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults

Background Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI <18·5 kg/m2) and obesity (BMI ≥30 kg/m2). For schoolaged children and adolescents, we report thinness (BMI <2 SD below the median of the WHO growth reference) and obesity (BMI >2 SD above the median). Findings From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and among boys in 15 countries (8%) with a posterior probability of at least 0·80, and increased among girls in 140 countries (70%) and boys in 137 countries (69%) with a posterior probability of at least 0·80. The countries with highest combined prevalence of thinness and obesity in school-aged children and adolescents in 2022 were in Polynesia and Micronesia and the Caribbean for both sexes, and Chile and Qatar for boys. Combined prevalence was also high in some countries in south Asia, such as India and Pakistan, where thinness remained prevalent despite having declined. In 2022, obesity in school-aged children and adolescents was more prevalent than thinness with a posterior probability of at least 0·80 among girls in 133 countries (67%) and boys in 125 countries (63%), whereas the converse was true in 35 countries (18%) and 42 countries (21%), respectively. In almost all countries for both adults and school-aged children and adolescents, the increases in double burden were driven by increases in obesity, and decreases in double burden by declining underweight or thinness. Interpretation The combined burden of underweight and obesity has increased in most countries, driven by an increase in obesity, while underweight and thinness remain prevalent in south Asia and parts of Africa. A healthy nutrition transition that enhances access to nutritious foods is needed to address the remaining burden of underweight while curbing and reversing the increase in obesit