Comparative analysis of toxic responses of organic extracts from diesel and selected alternative fuels engine emissions in human lung BEAS-2B cells

This study used toxicogenomics to identify the complex biological response of human lung BEAS-2B cells treated with organic components of particulate matter in the exhaust of a diesel engine. First, we characterized particles from standard diesel (B0), biodiesel (methylesters of rapeseed oil) in its neat form (B100) and 30% by volume blend with diesel fuel (B30), and neat hydrotreated vegetable oil (NEXBTL100). The concentration of polycyclic aromatic hydrocarbons (PAHs) and their derivatives in organic extracts was the lowest for NEXBTL100 and higher for biodiesel. We further analyzed global gene expression changes in BEAS-2B cells following 4 h and 24 h treatment with extracts. The concentrations of 50 mu g extract/mL induced a similar molecular response. The common processes induced after 4 h treatment included antioxidant defense, metabolism of xenobiotics and lipids, suppression of pro-apoptotic stimuli, or induction of plasminogen activating cascade; 24 h treatment affected fewer processes, particularly those involved in detoxification of xenobiotics, including PAHs. The majority of distinctively deregulated genes detected after both 4 h and 24 h treatment were induced by NEXBTL100; the deregulated genes included, e.g., those involved in antioxidant defense and cell cycle regulation and proliferation. B100 extract, with the highest PAH concentrations, additionally affected several cell cycle regulatory genes and p38 signaling.Web of Science1711art. no. 183

Inhalation of ZnO nanoparticles: Splice junction expression and alternative splicing in mice

Despite the wide application of nanomaterials, toxicity studies of nanoparticles (NP) are often limited to in vitro cell models, and the biological impact of NP exposure in mammals has not been thoroughly investigated. Zinc oxide (ZnO) NPs are commonly used in various consumer products. To evaluate the effects of the inhalation of ZnO NP in mice, we studied splice junction expression in the lungs as a proxy to gene expression changes analysis. Female ICR mice were treated with 6.46 x 10(4) and 1.93 x 10(6) NP/cm(3) for 3 days and 3 months, respectively. An analysis of differential expression and alternative splicing events in 298 targets (splice junctions) of 68 genes involved in the processes relevant to the biological effects of ZnO NP was conducted using next-generation sequencing. Three days of exposure resulted in the upregulation of IL-6 and downregulation of BID, GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junction expression; 3 months of exposure increased the expression of splice junctions in ALDH3A1, APAF1, BID, CASP3, DHCR7, GCLC, GCLM, GSR, GSS, EHHADH, FAS, HMOX-1, IFN, NF-kB1, NQO-1, PTGS1, PTGS2, RAD51, RIPK2, SRXN1, TRAF6, and TXNRD1. Alternative splicing of TRAF6 and TXNRD1 was induced after 3 days of exposure to 1.93 x 10(6) NP/cm(3). In summary, we observed changes of splice junction expression in genes involved in oxidative stress, apoptosis, immune response, inflammation, and DNA repair, as well as the induction of alternative splicing in genes associated with oxidative stress and inflammation. Our data indicate the potential negative biological effects of ZnO NP inhalation.Web of Science168120019

Influence of respirable fraction of suspended particulate matter in the air on functions of human cells' immunity system

Import 11/07/2012K posouzení imunomodulačních účinků vzorku reálných suspendovaných částic v ovzduší byly vybrány čtyři laboratorní imunologické testy umožňující hodnocení parametrů jak specifické, tak nespecifické imunitní odpovědi a alergické reakce. Testy byly prováděny v kulturách plné krve zdravých dobrovolných dárců. Výsledky imunologických testů neprokázaly vliv krátkodobých účinků suspendovaných částic v ovzduší na funkce buněk imunitního systému zdravých jedinců. Aplikace suspenze polétavého prachu neměla alergizační potenciál v testu degranulace bazofilů, nezvyšovala fagocytární aktivitu buněk v chemiluminiscenčním testu a nestimulovala proliferaci T lymfocytů v testu blastické transformace lymfocytů. Rovněž výsledky hodnocení produkce cytokinů multiplexovou metodou ALBIA ukazují, že testovaný vzorek polétavého prachu nepatří k silným induktorům funkcí imunitního systému.Immunomodulatory effects of a real airborne particulate sample were assessed using four laboratory immunoassays, allowing the evaluation of parameters of both the specific and nonspecific immune response and the allergy response. Assays were done in whole blood cultures from three healthy volunteers. The test results did not prove an influence of short-term effects of airborne particulate matter on immune cells´ functions of healthy persons. The presence of the aqueous suspension of particulate matter did not have an allergizing potential in the basophil degranulation assay, did not increase the phagocytic activity in the chemiluminescence assay and did not stimulate T-lymphocyte proliferation in the lymphocyte blast transformation assay. Likewise the results of the evaluation of cytokine production by a multiplex technology ALBIA show that the tested sample of airborne particulate matter does not belong to potent inductors of the immune system functions.Prezenční546 - Institut environmentálního inženýrstvívýborn

WHOLE-GENOME EXPRESSION ANALYSIS IN THP-1 MACROPHAGE-LIKE CELLS EXPOSED TO DIVERSE NANOMATERIALS

From the perspective of the immune system, nanomaterials (NMs) represent invading agents. Macrophages are immune cells residing in all organs and tissues as the first line of defense. Interactions of macrophages with NMs can determine the fate of NMs as well as their potential toxic effects. In the present study, we compared toxicity of four different types of NMs [NM-100 (TiO2, 110 nm), NM-110 (ZnO, 20 nm), NM-200 (SiO2, 150 nm) and NM-300K (Ag, 20 nm)], towards THP-1 macrophage-like cells. Cells were incubated with non-cytotoxic concentrations (1-25 mu g/ml) of NMs for 24 hours and microarray technology was used to analyze changes in whole-genome expression. Gene expression profiling revealed a substantially different molecular response following exposure to diverse NMs. While NM-100 did not exert any significant effect on gene expression profile, all other NMs triggered a pro-inflammatory response characterized by an activation of the NF-kappa B transcription factor and induced expression of numerous chemokines and cytokines. NM-110 and NM-300K further modulated processes such as DNA damage response, oxidative and replication stress as well as cell cycle progression and proteasome function. We suppose that genotoxicity of ZnO and Ag NMs leading to DNA damage and alternatively to apoptosis in THP-1 macrophages is probably caused by the extensive intracellular dissolution of these NPs, as confirmed by TEM imaging

Nanoparticle Safety Management with Regard to Occupational Safety

Nanomateriály (NMs) a jejich aplikace mohou významně přispět ke zlepšení kvality života i k řešení zásadních problémů, kterým čelí lidská společnost. Tytéž vlastnosti, které činí NMs unikátní a žádoucí, však mohou podmiňovat jejich toxicitu. Motivací práce bylo nalézt alespoň částečné řešení vedoucí k ochraně pracovníků v nanotechnologiích, kteří z důvodu vysokých koncentrací a dlouhodobé pravidelné expozice patří mezi osoby v největším ohrožení potenciálními nebezpečnými účinky NMs. V teoretické části práce byla analýzou použitelnosti standardních metod hodnocení rizik chemických látek na NMs zjištěna vysoká míra nejistot v oblasti charakterizace nebezpečnosti i hodnocení expozice v důsledku chybějících relevantních metod a spolehlivých experimentálních dat. Experimentální část práce se zabývá jak hodnocením známých mechanismů toxicity NMs, tak také nedořešenými toxikologickými otázkami, které byly detekovány v teoretické části práce: nekonzistentností experimentálních dat, vztahy mezi fyzikálně chemickými vlastnostmi a toxicitou, specifickými účinky NMs (zejména specifickými interakcemi NMs s imunitním systémem) a možnostmi jejich identifikace. Teoretické výsledky a praktické zkušenosti s testováním toxicity NMs byly využity při přípravě návrhu metodiky managementu rizik NMs. Jako vhodný vstupní nástroj byl vzhledem k vysokým nejistotám vyhodnocen kvalitativní přístup Control Banding (CB), spočívající v kategorizaci nebezpečnosti a expozice do tříd. Kombinace tříd expozice a nebezpečnosti udává třídu rizika, která určuje nutnost zavádění kontrolních a regulačních opatření. Byl vypracován přehled dostupných CB nástrojů pro NMs s ohledem na vhodnost jejich použití pro management rizik NMs v pracovním prostředí a vývojové schéma umožňující výběr optimálního nástroje pro konkrétní NM a podmínky expozice. Pro přesnější zařazení do třídy nebezpečnosti v rámci CB byla navržena baterie in vitro testů umožňující screeningové hodnocení možných toxických účinků NMs. Vybrané testy hodnotí čtyři endpointy, které odrážejí základní toxické účinky látek (cytotoxicitu, genotoxicitu) a známé nespecifické mechanismy toxicity typické pro NMs (oxidativní stres, chronický zánět). Baterie testů může být rovněž využita k porovnávání toxicity NMs s odlišnými fyzikálně-chemickými vlastnostmi a pro prioritizaci a případnou selekci méně toxických NMs již v ranné fázi jejich vývoje. Navržená strategie managementu rizik NMs má usnadnit zejména malým a středním podnikům implementaci adekvátních opatření pro zajištění bezpečnosti svých zaměstnanců.Nanomaterials (NMs) and their applications have the potential to significantly improve the quality of life as well as to contribute to solving the main challenges of today's society. However, the same properties that render NMs unique and beneficial could cause their enhanced or unexpected toxicity. The motivation for this thesis was to contribute to the protection of workers in nanotechnologies as they may be in a higher risk of potential adverse effects of NMs due to prolonged regular exposure and high exposure concentrations. Analysis of the applicability of the traditional chemical risk assessment approach for NMs revealed high levels of uncertainty in both hazard characterization and exposure assessment due to the lack of relevant standardized methods and reliable data. The experimental part of the thesis is focused on evaluation of known mechanisms of NMs toxicity as well as unresolved toxicological issues that were detected in the theoretical part: inconsistent experimental data, relationship between physico-chemical properties and toxicity of NMs, specific toxic effects of NMs (mainly interactions of NMs with the immune system) and methods for their identification. Theoretical findings and practical experience with toxicity testing of NMs were utilized to prepare a proposal of the NM risk management for occupational settings. Control Banding (CB), a qualitative approach, was selected as a suitable risk management tool with regard to the detected high uncertainties. CB categorizes hazard and exposure into different levels, referred to as bands. The combination of the hazard and exposure bands results into a risk band determining the necessity to implement control and regulatory measures. The available CB tools and their applicability for the occupational NM risk management were reviewed. Subsequently, a scheme for the selection of an optimal tool for particular NMs and specific exposure conditions was prepared. For more accurate hazard categorization, a set of in vitro toxicological assays enabling screening evaluation of potential toxic effects of NMs was proposed. The selected assays evaluate four endpoints covering basic toxic effects of substances (cytotoxicity, genotoxicity), as well as known non-specific mechanisms of toxicity typical for NMs (oxidative stress, inflammation). The testing battery can be used for the comparison of toxicity of NMs with different physico-chemical properties and to prioritize and select less toxic NMs in an early phase of their development. The proposed NM risk management strategy is intended to assist small and medium-sized enterprises to implement adequate measures to ensure the employee safety.040 - Katedra bezpečnosti práce a procesůvyhově

Nano-TiO2 stability in medium and size as important factors of toxicity in macrophage-like cells

TiO2 along with nano-TiO2 are commonly found in consumer products. In vivo studies have observed an accumulation of nano-TiO2 in macrophages. However, characteristics of nano-TiO2 determining toxicity remain unclear. In our study, the cytotoxic effects of 14 diverse nano-TiO2 on THP-1 macrophage-like cells were measured by 3 cytotoxicity assays (MTS, WST-1 and LDH). Total averaged cytotoxicity was calculated using principal component analysis. Characteristics of all 14 nano-TiO2 included hydrodynamic diameter, zeta potential, shape, polydispersity index (PDI) and concentration; moreover, crystal form, specific surface area and crystallite size were measured for 10 nano-TiO2. The variables affecting cytotoxicity were chosen using LASSO (least absolute shrinkage and selection operator). Except for concentration, PDI in media measured within 1 h after preparation of the nanomaterial dispersion was selected as a variable affecting cytotoxicity: stable dispersion resulted in higher cytotoxic effects. Crystallite size has been shown to have nonlinear effects (particles of sizes between 20 and 60 nm were cytotoxic while smaller and larger ones were not) and thus it has been excluded from LASSO. The shape (particles/fibre) and crystal form did not affect the cytotoxicity. PDI and the nonlinear effect of size could be an explanation for the inconsistencies of the cytotoxicity of nano-TiO2 in various studies.Web of Science5418817

Macrophage sensing of single-walled carbon nanotubes via Toll-like receptors

Abstract Carbon-based nanomaterials including carbon nanotubes (CNTs) have been shown to trigger inflammation. However, how these materials are ‘sensed’ by immune cells is not known. Here we compared the effects of two carbon-based nanomaterials, single-walled CNTs (SWCNTs) and graphene oxide (GO), on primary human monocyte-derived macrophages. Genome-wide transcriptomics assessment was performed at sub-cytotoxic doses. Pathway analysis of the microarray data revealed pronounced effects on chemokine-encoding genes in macrophages exposed to SWCNTs, but not in response to GO, and these results were validated by multiplex array-based cytokine and chemokine profiling. Conditioned medium from SWCNT-exposed cells acted as a chemoattractant for dendritic cells. Chemokine secretion was reduced upon inhibition of NF-κB, as predicted by upstream regulator analysis of the transcriptomics data, and Toll-like receptors (TLRs) and their adaptor molecule, MyD88 were shown to be important for CCL5 secretion. Moreover, a specific role for TLR2/4 was confirmed by using reporter cell lines. Computational studies to elucidate how SWCNTs may interact with TLR4 in the absence of a protein corona suggested that binding is guided mainly by hydrophobic interactions. Taken together, these results imply that CNTs may be ‘sensed’ as pathogens by immune cells