Last data update: Sep 16, 2024. (Total: 47680 publications since 2009)
Records 1-30 (of 66 Records) |
Query Trace: Shvedova A [original query] |
---|
Formation of protein adducts with Hydroperoxy-PE electrophilic cleavage products during ferroptosis
Amoscato AA , Anthonymuthu T , Kapralov O , Sparvero LJ , Shrivastava IH , Mikulska-Ruminska K , Tyurin VA , Shvedova AA , Tyurina YY , Bahar I , Wenzel S , Bayir H , Kagan VE . Redox Biol 2023 63 102758 Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process. |
Multi-walled carbon nanotubes elicit concordant changes in DNA methylation and gene expression following long-term pulmonary exposure in mice
Scala G , Delaval MN , Mukherjee SP , Federico A , Khaliullin TO , Yanamala N , Fatkhutdinova LM , Kisin ER , Greco D , Fadeel B , Shvedova AA . Carbon N Y 2021 178 563-572 Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) causes inflammation and fibrosis. Our previous work has shown that industrially produced MWCNTs trigger specific changes in gene expression in the lungs of exposed animals. To elucidate whether epigenetic effects play a role for these gene expression changes, we performed whole genome bisulphite sequencing to assess DNA methylation patterns in the lungs 56 days after exposure to MWCNTs. Lung tissues were also evaluated with respect to histopathological changes and cytokine profiling of bronchoalveolar lavage (BAL) fluid was conducted using a multi-plex array. Integrated analysis of transcriptomics data and DNA methylation data revealed concordant changes in gene expression. Functional analysis showed that the muscle contraction, immune system/inflammation, and extracellular matrix pathways were the most affected pathways. Taken together, the present study revealed that MWCNTs exert epigenetic effects in the lungs of exposed animals, potentially driving the subsequent gene expression changes. © 2021 The Authors |
Redox epiphospholipidome in programmed cell death signaling: Catalytic mechanisms and regulation
Kagan VE , Tyurina YY , Vlasova II , Kapralov AA , Amoscato AA , Anthonymuthu TS , Tyurin VA , Shrivastava IH , Cinemre FB , Lamade A , Epperly MW , Greenberger JS , Beezhold DH , Mallampalli RK , Srivastava AK , Bayir H , Shvedova AA . Front Endocrinol (Lausanne) 2020 11 628079 A huge diversification of phospholipids, forming the aqueous interfaces of all biomembranes, cannot be accommodated within a simple concept of their role as membrane building blocks. Indeed, a number of signaling functions of (phospho)lipid molecules has been discovered. Among these signaling lipids, a particular group of oxygenated polyunsaturated fatty acids (PUFA), so called lipid mediators, has been thoroughly investigated over several decades. This group includes oxygenated octadecanoids, eicosanoids, and docosanoids and includes several hundreds of individual species. Oxygenation of PUFA can occur when they are esterified into major classes of phospholipids. Initially, these events have been associated with non-specific oxidative injury of biomembranes. An alternative concept is that these post-synthetically oxidatively modified phospholipids and their adducts with proteins are a part of a redox epiphospholipidome that represents a rich and versatile language for intra- and inter-cellular communications. The redox epiphospholipidome may include hundreds of thousands of individual molecular species acting as meaningful biological signals. This review describes the signaling role of oxygenated phospholipids in programs of regulated cell death. Although phospholipid peroxidation has been associated with almost all known cell death programs, we chose to discuss enzymatic pathways activated during apoptosis and ferroptosis and leading to peroxidation of two phospholipid classes, cardiolipins (CLs) and phosphatidylethanolamines (PEs). This is based on the available LC-MS identification and quantitative information on the respective peroxidation products of CLs and PEs. We focused on molecular mechanisms through which two proteins, a mitochondrial hemoprotein cytochrome c (cyt c), and non-heme Fe lipoxygenase (LOX), change their catalytic properties to fulfill new functions of generating oxygenated CL and PE species. Given the high selectivity and specificity of CL and PE peroxidation we argue that enzymatic reactions catalyzed by cyt c/CL complexes and 15-lipoxygenase/phosphatidylethanolamine binding protein 1 (15LOX/PEBP1) complexes dominate, at least during the initiation stage of peroxidation, in apoptosis and ferroptosis. We contrast cell-autonomous nature of CLox signaling in apoptosis correlating with its anti-inflammatory functions vs. non-cell-autonomous ferroptotic signaling facilitating pro-inflammatory (necro-inflammatory) responses. Finally, we propose that small molecule mechanism-based regulators of enzymatic phospholipid peroxidation may lead to highly specific anti-apoptotic and anti-ferroptotic therapeutic modalities. |
Differential responses of murine alveolar macrophages to elongate mineral particles of asbestiform and non-asbestiform varieties: Cytotoxicity, cytokine secretion and transcriptional changes.
Khaliullin TO , Kisin ER , Guppi S , Yanamala N , Zhernovkov V , Shvedova AA . Toxicol Appl Pharmacol 2020 409 115302 Human exposures to asbestiform elongate mineral particles (EMP) may lead to diffuse fibrosis, lung cancer, malignant mesothelioma and autoimmune diseases. Cleavage fragments (CF) are chemically identical to asbestiform varieties (or habits) of the parent mineral, but no consensus exists on whether to treat them as asbestos from toxicological and regulatory standpoints. Alveolar macrophages (AM) are the first responders to inhaled particulates, participating in clearance and activating other resident and recruited immunocompetent cells, impacting the long-term outcomes. In this study we address how EMP of asbestiform versus non-asbestiform habit affect AM responses. Max Planck Institute (MPI) cells, a non-transformed mouse line that has an AM phenotype and genotype, were treated with mass-, surface area- (s.a.), and particle number- (p.n.) equivalent concentrations of respirable asbestiform and non-asbestiform riebeckite/tremolite EMP for 24 h. Cytotoxicity, cytokines secretion and transcriptional changes were evaluated. At the equal mass, asbestiform EMP were more cytotoxic, however EMP of both habits induced similar LDH leakage and decrease in viability at s.a. and p.n. equivalent doses. DNA damage assessment and cell cycle analysis revealed differences in the modes of cell death between asbestos and respective CF. There was an increase in chemokines, but not pro-inflammatory cytokines after all EMP treatments. Principal component analysis of the cytokine secretion showed close clustering for the s.a. and p.n. equivalent treatments. There were mineral- and habit-specific patterns of gene expression dysregulation at s.a. equivalent doses. Our study reveals the critical nature of EMP morphometric parameters for exposure assessment and dosing approaches used in toxicity studies. |
Enhanced morphological transformation of human lung epithelial cells by continuous exposure to cellulose nanocrystals
Kisin ER , Yanamala N , Rodin D , Menas A , Farcas M , Russo M , Guppi S , Khaliullin TO , Iavicoli I , Harper M , Star A , Kagan VE , Shvedova AA . Chemosphere 2020 250 126170 Cellulose nanocrystals (CNC), also known as nanowhiskers, have recently gained much attention due to their biodegradable nature, advantageous chemical and mechanical properties, economic value and renewability thus making them attractive for a wide range of applications. However, before these materials can be considered for potential uses, investigation of their toxicity is prudent. Although CNC exposures are associated with pulmonary inflammation and damage as well as oxidative stress responses and genotoxicity in vivo, studies evaluating cell transformation or tumorigenic potential of CNC's were not previously conducted. In this study, we aimed to assess the neoplastic-like transformation potential of two forms of CNC derived from wood (powder and gel) in human pulmonary epithelial cells (BEAS-2B) in comparison to fibrous tremolite (TF), known to induce lung cancer. Short-term exposure to CNC or TF induced intracellular ROS increase and DNA damage while long-term exposure resulted in neoplastic-like transformation demonstrated by increased cell proliferation, anchorage-independent growth, migration and invasion. The increased proliferative responses were also in-agreement with observed levels of pro-inflammatory cytokines. Based on the hierarchical clustering analysis (HCA) of the inflammatory cytokine responses, CNC powder was segregated from the control and CNC-gel samples. This suggests that CNC may have the ability to influence neoplastic-like transformation events in pulmonary epithelial cells and that such effects are dependent on the type/form of CNC. Further studies focusing on determining and understanding molecular mechanisms underlying potential CNC cell transformation events and their likelihood to induce tumorigenic effects in vivo are highly warranted. |
Comparative analysis of lung and blood transcriptomes in mice exposed to multi-walled carbon nanotubes
Khaliullin TO , Yanamala N , Newman MS , Kisin ER , Fatkhutdinova LM , Shvedova AA . Toxicol Appl Pharmacol 2020 390 114898 Pulmonary exposure to multi-walled carbon nanotubes (MWCNT) causes inflammation, fibroproliferation, immunotoxicity, and systemic responses in rodents. However, the search for representative biomarkers of exposure is an ongoing endeavor. Whole blood gene expression profiling is a promising new approach for the identification of novel disease biomarkers. We asked if the whole blood transcriptome reflects pathology-specific changes in lung gene expression caused by MWCNT. To answer this question, we performed mRNA sequencing analysis of the whole blood and lung in mice administered MWCNT or vehicle solution via pharyngeal aspiration and sacrificed 56days later. The pattern of lung mRNA expression as determined using Ingenuity Pathway Analysis (IPA) was indicative of continued inflammation, immune cell trafficking, phagocytosis, and adaptive immune responses. Simultaneously, innate immunity-related transcripts (Plunc, Bpifb1, Reg3g) and cancer-related pathways were downregulated. IPA analysis of the differentially expressed genes in the whole blood suggested increased hematopoiesis, predicted activation of cancer/tumor development pathways, and atopy. There were several common upregulated genes between whole blood and lungs, important for adaptive immune responses: Cxcr1, Cd72, Sharpin, and Slc11a1. Trim24, important for TH2 cell effector function, was downregulated in both datasets. Hla-dqa1 mRNA was upregulated in the lungs and downregulated in the blood, as was Lilrb4, which controls the reactivity of immune response. "Cancer" disease category had opposing activation status in the two datasets, while the only commonality was "hypersensitivity". Transcriptome changes occurring in the lungs did not produce a completely replicable pattern in whole blood; however, specific systemic responses may be shared between transcriptomic profiles. |
Redox phospholipidomics of enzymatically generated oxygenated phospholipids as specific signals of programmed cell death
Kagan VE , Tyurina YY , Sun WY , Vlasova II , Dar H , Tyurin VA , Amoscato AA , Mallampalli R , van der Wel PCA , He RR , Shvedova AA , Gabrilovich DI , Bayir H . Free Radic Biol Med 2019 147 231-241 High fidelity and effective adaptive changes of the cell and tissue metabolism to changing environments require strict coordination of numerous biological processes. Multicellular organisms developed sophisticated signaling systems of monitoring and responding to these different contexts. Among these systems, oxygenated lipids play a significant role realized via a variety of re-programming mechanisms. Some of them are enacted as a part of pro-survival pathways that eliminate harmful or unnecessary molecules or organelles by a variety of degradation/hydrolytic reactions or specialized autophageal processes. When these "partial" intracellular measures are insufficient, the programs of cells death are triggered with the aim to remove irreparably damaged members of the multicellular community. These regulated cell death mechanisms are believed to heavily rely on signaling by a highly diversified group of molecules, oxygenated phospholipids (PLox). Out of thousands of detectable individual PLox species, redox phospholipidomics deciphered several specific molecules that seem to be diagnostic of specialized death programs. Oxygenated cardiolipins (CLs) and phosphatidylethanolamines (PEs) have been identified as predictive biomarkers of apoptosis and ferroptosis, respectively. This has led to decoding of the enzymatic mechanisms of their formation involving mitochondrial oxidation of CLs by cytochrome c and endoplasmic reticulum-associated oxidation of PE by lipoxygenases. Understanding of the specific biochemical radical-mediated mechanisms of these oxidative reactions opens new avenues for the design and search of highly specific regulators of cell death programs. This review emphasizes the usefulness of such selective lipid peroxidation mechanisms in contrast to the concept of random poorly controlled free radical reactions as instruments of non-specific damage of cells and their membranes. Detailed analysis of two specific examples of phospholipid oxidative signaling in apoptosis and ferroptosis along with their molecular mechanisms and roles in reprogramming has been presented. |
Redox (phospho)lipidomics of signaling in inflammation and programmed cell death
Tyurina YY , St Croix CM , Watkins SC , Watson AM , Epperly MW , Anthonymuthu TS , Kisin ER , Vlasova II , Krysko O , Krysko DV , Kapralov AA , Dar HH , Tyurin VA , Amoscato AA , Popova EN , Bolevich SB , Timashev PS , Kellum JA , Wenzel SE , Mallampalli RK , Greenberger JS , Bayir H , Shvedova AA , Kagan VE . J Leukoc Biol 2019 106 (1) 57-81 In addition to the known prominent role of polyunsaturated (phospho)lipids as structural blocks of biomembranes, there is an emerging understanding of another important function of these molecules as a highly diversified signaling language utilized for intra- and extracellular communications. Technological developments in high-resolution mass spectrometry facilitated the development of a new branch of metabolomics, redox lipidomics. Analysis of lipid peroxidation reactions has already identified specific enzymatic mechanisms responsible for the biosynthesis of several unique signals in response to inflammation and regulated cell death programs. Obtaining comprehensive information about millions of signals encoded by oxidized phospholipids, represented by thousands of interactive reactions and pleiotropic (patho)physiological effects, is a daunting task. However, there is still reasonable hope that significant discoveries, of at least some of the important contributors to the overall overwhelmingly complex network of interactions triggered by inflammation, will lead to the discovery of new small molecule regulators and therapeutic modalities. For example, suppression of the production of AA-derived pro-inflammatory mediators, HXA3 and LTB4, by an iPLA2 gamma inhibitor, R-BEL, mitigated injury associated with the activation of pro-inflammatory processes in animals exposed to whole-body irradiation. Further, technological developments promise to make redox lipidomics a powerful approach in the arsenal of diagnostic and therapeutic instruments for personalized medicine of inflammatory diseases and conditions. |
Comparative cytotoxicity of respirable surface-treated/untreated calcium carbonate rock dust particles in vitro
Khaliullin TO , Kisin ER , Yanamala N , Guppi S , Harper M , Lee T , Shvedova AA . Toxicol Appl Pharmacol 2018 362 67-76 Calcium carbonate rock dust (RD) is used in mining to reduce the explosivity of aerosolized coal. During the dusting procedures, potential for human exposure occurs, raising health concerns. To improve RD aerosolization, several types of anti-caking surface treatments exist. The aim of the study was to evaluate cytotoxicity of four respirable RD samples: untreated/treated limestone (UL/TL), untreated/treated marble (UM/TM), and crystalline silica (SiO2) as a positive control in A549 and THP-1 transformed human cell lines. Respirable fractions were generated and collected using FSP10 high flow-rate cyclone samplers. THP-1 cells were differentiated with phorbol-12-myristate-13-acetate (20ng/ml, 48h). Cells were exposed to seven different concentrations of RD and SiO2 (0-0.2mg/ml). RD caused a slight decrease in viability at 24 or 72h post-exposure and were able to induce inflammatory cytokine production in A549 cells, however, with considerably less potency than SiO2. In THP-1 cells at 24h, there was significant dose-dependent lactate dehydrogenase, inflammatory cytokine and chemokine release. Caspase-1 activity was increased in SiO2- and, on a lesser scale, in TM- exposed cells. To test if the increased toxicity of TM was uptake-related, THP-1 cells were pretreated with Cytochalasin D (CytD) or Bafilomycin A (BafA), followed by exposure to RD or SiO2 for 6h. CytD blocked the uptake and significantly decreased cytotoxicity of all particles, while BafA prevented caspase-1 activation but not cytotoxic effects of TM. Only TM was able to induce an inflammatory response in THP-1 cells, however it was much less pronounced compared to silica. |
"Only a life lived for others is worth living": Redox signaling by oxygenated phospholipids in cell fate decisions
Tyurina YY , Shrivastava I , Tyurin VA , Mao G , Dar HH , Watkins S , Epperly M , Bahar I , Shvedova AA , Pitt B , Wenzel SE , Mallampalli RK , Sadovsky Y , Gabrilovich D , Greenberger JS , Bayir H , Kagan VE . Antioxid Redox Signal 2018 29 (13) 1333-1358 SIGNIFICANCE: Oxygenated polyunsaturated lipids are known to play multi-functional roles as essential signals coordinating metabolism and physiology. Among them are well-studied eicosanoids and docosanoids that are generated via phospholipase A2 hydrolysis of membrane phospholipids and subsequent oxygenation of free polyunsaturated fatty acids (PUFA) by cyclooxygenases and lipoxygenases. Recent Advances: There is an emerging understanding that oxygenated PUFA-phospholipids also represent a rich signaling language with yet-to-be-deciphered details of the execution machinery-oxygenating enzymes, regulators, and receptors. Both free and esterified oxygenated PUFA signals are generated in cells, and their cross-talk and inter-conversion through the de-acylation/re-acylation reactions is not sufficiently explored. CRITICAL ISSUES: Here, we review recent data related to oxygenated phospholipids as important damage signals that trigger programmed cell death pathways to eliminate irreparably injured cells and preserve the health of multicellular environments. We discuss the mechanisms underlying the trans-membrane redistribution and generation of oxygenated cardiolipins in mitochondria by cytochrome c as pro-apoptotic signals. We also consider the role of oxygenated phosphatidylethanolamines as proximate pro-ferroptotic signals. FUTURE DIRECTIONS: We highlight the importance of sequential processes of phospholipid oxygenation and signaling in disease contexts as opportunities to use their regulatory mechanisms for the identification of new therapeutic targets. |
Iron catalysis of lipid peroxidation in ferroptosis: Regulated enzymatic or random free radical reaction
Stoyanovsky DA , Tyurina YY , Shrivastava I , Bahar I , Tyurin VA , Protchenko O , Jadhav S , Bolevich SB , Kozlov AV , Vladimirov YA , Shvedova AA , Philpott CC , Bayir H , Kagan VE . Free Radic Biol Med 2018 133 153-161 Duality of iron as an essential cofactor of many enzymatic metabolic processes and as a catalyst of poorly controlled redox-cycling reactions defines its possible biological beneficial and hazardous role in the body. In this review, we discuss these two "faces" of iron in a newly conceptualized program of regulated cell death, ferroptosis. Ferroptosis is a genetically programmed iron-dependent form of regulated cell death driven by enhanced lipid peroxidation and insufficient capacity of thiol-dependent mechanisms (glutathione peroxidase 4, GPX4) to eliminate hydroperoxy-lipids. We present arguments favoring the enzymatic mechanisms of ferroptotically engaged non-heme iron of 15-lipoxygenases (15-LOX) in complexes with phosphatidylethanolamine binding protein 1 (PEBP1) as a catalyst of highly selective and specific oxidation reactions of arachidonoyl- (AA) and adrenoyl-phosphatidylethanolamines (PE). We discuss possible role of iron chaperons as control mechanisms for guided iron delivery directly to their "protein clients" thus limiting non-enzymatic redox-cycling reactions. We also consider opportunities of loosely-bound iron to contribute to the production of pro-ferroptotic lipid oxidation products. Finally, we propose a two-stage iron-dependent mechanism for iron in ferroptosis by combining its catalytic role in the 15-LOX-driven production of 15-hydroperoxy-AA-PE (HOO-AA-PE) as well as possible involvement of loosely-bound iron in oxidative cleavage of HOO-AA-PE to oxidatively truncated electrophiles capable of attacking nucleophilic targets in yet to be identified proteins leading to cell demise. |
Fibrous nanocellulose, crystalline nanocellulose, carbon nanotubes, and crocidolite asbestos elicit disparate immune responses upon pharyngeal aspiration in mice
Park EJ , Khaliullin TO , Shurin MR , Kisin ER , Yanamala N , Fadeel B , Chang J , Shvedova AA . J Immunotoxicol 2018 15 (1) 12-23 With the rapid development of synthetic alternatives to mineral fibers, their possible effects on the environment and human health have become recognized as important issues worldwide. This study investigated effects of four fibrous materials, i.e. nanofibrillar/nanocrystalline celluloses (NCF and CNC), single-walled carbon nanotubes (CNTs), and crocidolite asbestos (ASB), on pulmonary inflammation and immune responses found in the lungs, as well as the effects on spleen and peripheral blood immune cell subsets. BALB/c mice were given NCF, CNC, CNT, and ASB on Day 1 by oropharyngeal aspiration. At 14 days post-exposure, the animals were evaluated. Total cell number, mononuclear phagocytes, polymorphonuclear leukocytes, lymphocytes, and LDH levels were significantly increased in ASB and CNT-exposed mice. Expression of cytokines and chemokines in bronchoalveolar lavage (BAL) was quite different in mice exposed to four particle types, as well as expression of antigen presentation-related surface proteins on BAL cells. The results revealed that pulmonary exposure to fibrous materials led to discrete local immune cell polarization patterns with a TH2-like response caused by ASB and TH1-like immune reaction to NCF, while CNT and CNC caused non-classical or non-uniform responses. These alterations in immune response following pulmonary exposure should be taken into account when testing the applicability of new nanosized materials with fibrous morphology. |
Macrophage sensing of single-walled carbon nanotubes via Toll-like receptors
Mukherjee SP , Bondarenko O , Kohonen P , Andon FT , Brzicova T , Gessner I , Mathur S , Bottini M , Calligari P , Stella L , Kisin E , Shvedova A , Autio R , Salminen-Mankonen H , Lahesmaa R , Fadeel B . Sci Rep 2018 8 (1) 1115 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-kappaB, 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. |
Characterization of pulmonary responses in mice to asbestos/asbestiform fibers using gene expression profiles.
Yanamala N , Kisin ER , Gutkin DW , Shurin MR , Harper M , Shvedova AA . J Toxicol Environ Health A 2018 81 (4) 60-79 Humans exposed to asbestos and/or asbestiform fibers are at high risk of developing many lung diseases including asbestosis, lung cancer, and malignant mesothelioma. However, the disease-causing potential and specific metabolic mechanisms and pathways associated with various asbestos/asbestiform fiber exposures triggering different carcinogenic and non-carcinogenic outcomes are still largely unknown. The aim of this this study was to investigate gene expression profiles and inflammatory responses to different asbestos/asbestiform fibers at the acute/sub-acute phase that may be related to delayed pathological outcomes observed at later time points. Mice were exposed to asbestos (crocidolite, tremolite asbestos), asbestiform fibers (erionite), and a low pathogenicity mineral fiber (wollastonite) using oropharyngeal aspiration. Similarities in inflammatory and tissue damage responses, albeit with quantitative differences, were observed at day 1 and 7 post treatment. Exposure to different fibers induced significant changes in regulation and release of a number of inflammatory cytokines/chemokines. Comparative analysis of changes in gene regulation in the lung on day 7 post exposure were interpretable in the context of differential biological responses that were consistent with histopathological findings at days 7 and 56 post treatment. Our results noted differences in the magnitudes of pulmonary responses and gene regulation consistent with pathological alterations induced by exposures to four asbestos/asbestiform fibers examined. Further comparative mechanistic studies linking early responses with the long-term endpoints may be instrumental to understanding triggering mechanisms underlying pulmonary carcinogenesis, that is lung cancer versus mesothelioma. |
Mediation of the single-walled carbon nanotubes induced pulmonary fibrogenic response by osteopontin and TGF-beta1
Khaliullin TO , Kisin ER , Murray AR , Yanamala N , Shurin MR , Gutkin DW , Fatkhutdinova LM , Kagan VE , Shvedova AA . Exp Lung Res 2017 43 (8) 311-326 PURPOSE OF THE STUDY: A number of in vivo studies have shown that pulmonary exposure to carbon nanotubes (CNTs) may lead to an acute local inflammatory response, pulmonary fibrosis, and granulomatous lesions. Among the factors that play direct roles in initiation and progression of fibrotic processes are epithelial-mesenchymal transition and myofibroblasts recruitment/differentiation, both mediated by transforming growth factor-beta1 (TGF-beta1). Yet, other contributors to TGF-beta1 associated signaling, such as osteopontin (OPN) has not been fully investigated. MATERIALS AND METHODS: OPN-knockout female mice (OPN-KO) along with their wild-type (WT) counterparts were exposed to single-walled carbon nanotubes (SWCNT) (40 microg/mouse) via pharyngeal aspiration and fibrotic response was assessed 1, 7, and 28 days post-exposure. Simultaneously, RAW 264.7 and MLE-15 cells were treated with SWCNT (24 hours, 6 microg/cm(2) to 48 microg/cm(2)) or bleomycin (0.1 microg/ml) in the presence of OPN-blocking antibody or isotype control, and TGF-beta1 was measured in supernatants. RESULTS AND CONCLUSIONS: Diminished lactate dehydrogenase activity at all time points, along with less pronounced neutrophil influx 24 h post-exposure, were measured in broncho-alveolar lavage (BAL) of OPN-KO mice compared to WT. Pro-inflammatory cytokine release (IL-6, TNF-alpha, MCP-1) was reduced. A significant two-fold increase of TGF-beta1 was found in BAL of WT mice at 7 days, while TGF-beta1 levels in OPN-KO animals remained unaltered. Histological examination revealed marked decrease in granuloma formation and less collagen deposition in the lungs of OPN-KO mice compared to WT. RAW 264.7 but not MLE-15 cells exposed to SWCNT and bleomycin had significantly less TGF-beta1 released in the presence of OPN-blocking antibody. We believe that OPN is important in initiating the cellular mechanisms that produce an overall pathological response to SWCNT and it may act upstream of TGF-beta1. Further investigation to understand the mechanistic details of such interactions is critical to predict outcomes of pulmonary exposure to CNT. |
Ins and outs in environmental and occupational safety studies of asthma and engineered nanomaterials
Dobrovolskaia MA , Shurin MR , Kagan VE , Shvedova AA . ACS Nano 2017 11 (8) 7565-7571 According to the Centers for Disease Control and Prevention, approximately 25 million Americans suffer from asthma. The disease total annual cost is about $56 billion and includes both the direct and indirect costs of medications, hospital stays, missed work, and decreased productivity. Air pollution with xenobiotics, bacterial agents, and industrial nanomaterials, such as carbon nanotubes, contribute to the exacerbation of this condition and are a point of particular attention in environmental toxicology as well as in occupational health and safety research. Mast cell degranulation and activation of Th2 cells triggered either by allergen-specific immunoglobulin E (IgE) or by alternative mechanisms, such as locally produced neurotransmitters, underlie the pathophysiological process of airway constriction during an asthma attack. Other immune and non-immune cell types, including basophils, eosinophils, Th1, Th17, Th9, macrophages, dendritic cells, and smooth muscle cells, are involved in the inflammatory and allergic responses during asthma, which, under chronic conditions, may progress without mast cells, the key trigger of the acute asthma attack. To decipher complex molecular, cellular, and genetic mechanisms, many researchers have attempted to develop in vitro and in vivo models to study asthma. Herein, we summarize the advantages and disadvantages of various models and their applicability to nanoparticle evaluation in asthma research. We further suggest that a framework for both in vitro and in vivo methods should be used to study the impact of engineered nanomaterials on asthma etiology, pathophysiology, and treatment. |
Nanotechnology in agriculture: Opportunities, toxicological implications, and occupational risks
Iavicoli I , Leso V , Beezhold DH , Shvedova AA . Toxicol Appl Pharmacol 2017 329 96-111 Nanotechnology has the potential to make a beneficial impact on several agricultural, forestry, and environmental challenges, such as urbanization, energy constraints, and sustainable use of resources. However, new environmental and human health hazards may emerge from nano-enhanced applications. This raises concerns for agricultural workers who may become primarily exposed to such xenobiotics during their job tasks. The aim of this review is to discuss promising solutions that nanotechnology may provide in agricultural activities, with a specific focus on critical aspects, challenging issues, and research needs for occupational risk assessment and management in this emerging field. Eco-toxicological aspects were not the focus of the review. Nano-fertilizers, (nano-sized nutrients, nano-coated fertilizers, or engineered metal-oxide or carbon-based nanomaterials per se), and nano-pesticides, (nano-formulations of traditional active ingredients or inorganic nanomaterials), may provide a targeted/controlled release of agrochemicals, aimed to obtain their fullest biological efficacy without over-dosage. Nano-sensors and nano-remediation methods may detect and remove environmental contaminants. However, limited knowledge concerning nanomaterial biosafety, adverse effects, fate, and acquired biological reactivity once dispersed into the environment, requires further scientific efforts to assess possible nano-agricultural risks. In this perspective, toxicological research should be aimed to define nanomaterial hazards and levels of exposure along the life-cycle of nano-enabled products, and to assess those physico-chemical features affecting nanomaterial toxicity, possible interactions with agro-system co-formulants, and stressors. Overall, this review highlights the importance to define adequate risk management strategies for workers, occupational safety practices and policies, as well as to develop a responsible regulatory consensus on nanotechnology in agriculture. |
Fibrillar vs crystalline nanocellulose pulmonary epithelial cell responses: Cytotoxicity or inflammation?
Menas AL , Yanamala N , Farcas MT , Russo M , Friend S , Fournier PM , Star A , Iavicoli I , Shurin GV , Vogel UB , Fadeel B , Beezhold D , Kisin ER , Shvedova AA . Chemosphere 2016 171 671-680 Nanocellulose (NC) is emerging as a highly promising nanomaterial for a wide range of applications. Moreover, many types of NC are produced, each exhibiting a slightly different shape, size, and chemistry. The main objective of this study was to compare cytotoxic effects of cellulose nanocrystals (CNC) and nanofibrillated cellulose (NCF). The human lung epithelial cells (A549) were exposed for 24 h and 72 h to five different NC particles to determine how variations in properties contribute to cellular outcomes, including cytotoxicity, oxidative stress, and cytokine secretion. Our results showed that NCF were more toxic compared to CNC particles with respect to cytotoxicity and oxidative stress responses. However, exposure to CNC caused an inflammatory response with significantly elevated inflammatory cytokines/chemokines compared to NCF. Interestingly, cellulose staining indicated that CNC particles, but not NCF, were taken up by the cells. Furthermore, clustering analysis of the inflammatory cytokines revealed a similarity of NCF to the carbon nanofibers response and CNC to the chitin, a known immune modulator and innate cell activator. Taken together, the present study has revealed distinct differences between fibrillar and crystalline nanocellulose and demonstrated that physicochemical properties of NC are critical in determining their toxicity. |
Hollow carbon spheres trigger inflammasome-dependent IL-1beta secretion in macrophages
Andon FT , Mukherjee SP , Gessner I , Wortmann L , Xiao L , Hultenby K , Shvedova AA , Mathur S , Fadeel B . Carbon N Y 2017 113 243-251 It is disputed whether inflammasome activation leading to secretion of pro-inflammatory interleukin (IL)-1beta in macrophages transpires independently of cell death or whether the two processes are linked. Here, we synthesized hollow carbon spheres (HCS) and investigated their effects on primary human monocyte-derived macrophages (HMDM); short (500 nm) non-functionalized single-walled carbon nanotubes (SWCNT) were included for comparison. HCS (250 nm) were readily taken up by HMDM and induced ROS production, but did not trigger a loss of cell viability. However, a dose- and time-dependent release of IL-1beta was detected in lipopolysaccharide (LPS)-primed macrophages upon exposure to HCS, while SWCNT-induced secretion of IL-1β was less pronounced. HCS-triggered IL-1beta secretion was cathepsin B- and caspase-1-dependent, and was accompanied by a reduction in intracellular K+. Furthermore, cytokine secretion was reduced following treatment with the antioxidant, N-acetylcysteine, and cytochalasin D, an inhibitor of actin polymerization. HCS also triggered IL-1beta release in LPS-primed THP.1 cells, but not in THP.1 cells with silencing of ASC, NLRP3, or caspase-1 expression, providing evidence that IL-1beta was elicited through NLRP3 inflammasome activation. These studies shed light on the effects of HCS on primary macrophages, and show that spherical carbon-based nanoparticles are potent inflammasome activators. |
In vitro toxicity evaluation of lignin-(un)coated cellulose based nanomaterials on human A549 and THP-1 cells
Yanamala N , Kisin ER , Menas AL , Farcas MT , Khaliullin TO , Vogel U , Shurin GV , Schwegler-Berry D , Fournier PM , Star A , Shvedova AA . Biomacromolecules 2016 17 (11) 3464-3473 A significant amount of research towards commercial development of cellulose based nanomaterials (CNM) is now in progress with some potential applications. Using human A549 and THP-1 cells, we evaluated the biological responses of various CNMs, made out of similar material but with functional and morphological variations. While A549 cells displayed minimal or no cytotoxic responses following exposure to CNMs, THP-1 cells were more susceptible to cytotoxicity, cellular damage and inflammatory responses. Further analysis of these biological responses evaluated using hierarchical clustering approaches was effective in discriminating (dis)-similarities of various CNMs studied and identified potential inflammatory factors contributing to cytotoxicity. No correlation between cytotoxicity and surface properties of CNMs was found. This study clearly highlights that in addition to the source and characteristics of CNMs, cell type-specific differences in the recognition/uptake of CNMs along with their inherent capability to respond to external stimuli, are crucial for assessing the toxicity of CNMs. |
Pulmonary exposure to cellulose nanocrystals caused deleterious effects to reproductive system in male mice
Farcas MT , Kisin ER , Menas AL , Gutkin DW , Star A , Reiner RS , Yanamala N , Savolainen K , Shvedova AA . J Toxicol Environ Health A 2016 79 (21) 1-14 Over the past several years there has been an increased number of applications of cellulosic materials in many sectors, including the food industry, cosmetics, and pharmaceuticals. However, to date, there are few studies investigating the potential adverse effects of cellulose nanocrystals (CNC). The objective of this study was to determine long-term outcomes on the male reproductive system of mice upon repeated pharyngeal aspiration exposure to CNC. To achieve this, cauda epididymal sperm samples were analyzed for sperm concentration, motility, morphological abnormalities, and DNA damage. Testicular and epididymal oxidative damage was evaluated, as well as histopathology examination of testes. In addition, changes in levels of testosterone in testes and serum and of luteinizing hormone (LH) in serum were determined. Three months after the last administration, CNC exposure significantly altered sperm concentration, motility, cell morphology, and sperm DNA integrity. These parameters correlated with elevated proinflammatory cytokines levels and myeloperoxidase (MPO) activity in testes, as well as oxidative stress in both testes and epididymis. Exposure to CNC also produced damage to testicular structure, as evidenced by presence of interstitial edema, frequent dystrophic seminiferous tubules with arrested spermatogenesis and degenerating spermatocytes, and imbalance in levels of testosterone and LH. Taken together, these results demonstrate that pulmonary exposure to CNC induces sustained adverse effects in spermatocytes/spermatozoa, suggesting male reproductive toxicity. |
Gender differences in murine pulmonary responses elicited by cellulose nanocrystals
Shvedova AA , Kisin ER , Yanamala N , Farcas MT , Menas AL , Williams A , Fournier PM , Reynolds JS , Gutkin DW , Star A , Reiner RS , Halappanavar S , Kagan VE . Part Fibre Toxicol 2016 13 (1) 28 BACKGROUND: Cellulose-based materials have been used for centuries to manufacture different goods derived from forestry and agricultural sources. In the growing field of nanocellulose applications, its uniquely engineered properties are instrumental for inventive products coming to competitive markets. Due to their high aspect ratio and stiffness, it is speculated that cellulose nanocrystals (CNC) may cause similar pulmonary toxicity as carbon nanotubes and asbestos, thus posing a potential negative impact on public health and the environment. METHODS: The present study was undertaken to investigate the pulmonary outcomes induced by repeated exposure to respirable CNC. C57BL/6 female and male mice were exposed by pharyngeal aspiration to CNC (40 mug/mouse) 2 times a week for 3 weeks. Several biochemical endpoints and pathophysiological outcomes along with gene expression changes were evaluated and compared in the lungs of male and female mice. RESULTS: Exposure to respirable CNC caused pulmonary inflammation and damage, induced oxidative stress, elevated TGF-beta and collagen levels in lung, and impaired pulmonary functions. Notably, these effects were markedly more pronounced in females compared to male mice. Moreover, sex differences in responses to pulmonary exposure to CNC were also detected at the level of global mRNA expression as well as in inflammatory cytokine/chemokine activity. CONCLUSIONS: Overall, our results indicate that there are considerable differences in responses to respirable CNC based on gender with a higher pulmonary toxicity observed in female mice. |
Assessment of airborn multiwalled carbon nanotubes in a manufactoring environment
Fatkhutdinova LM , Khaliullin TO , Zalyalov RR , Tkachev AG , Birch ME , Shvedova AA . Nanotechnol Russ 2016 11 (1) 110-116 This study was carried out in a factory producing multiwalled carbon nanotubes (MWCNTs) by the catalytic chemical vapor deposition method in a pyrolysis reactor. Air samples of the personal breathing areas were collected simultaneously on mixed cellulose ester filters, for analysis by transmission electron s (TEM), and on high-purity quartz filters for thermal-optical analysis of elemental carbon (EC). It is found that the production of MWCNTs is accompanied by the release of the MWCNT structures in the air of different working zones. The concentration of respirable aerosol in the personal breathing areas, averaged over an 8-hour period, ranges from 0.54 to 6.11 μg/m3 based on EC. Airborne MWCNTs were found in the form of agglomerates that range in size from about 1 to 10 μm. These data are consistent with measurements in different plants by two other international groups (from the United States and Sweden) using similar methodology (TEM in combination with EC analysis). In the absence of convincing data on the potential health risks of MWCNTs, and following the principle of reasonable precautions, preventive measures should be taken to minimize exposure to these materials. |
Applied Nanotoxicology
Hobson DW , Roberts SM , Shvedova AA , Warheit DB , Hinkley GK , Guy RC . Int J Toxicol 2016 35 (1) 5-16 Nanomaterials, including nanoparticles and nanoobjects, are being incorporated into everyday products at an increasing rate. These products include consumer products of interest to toxicologists such as pharmaceuticals, cosmetics, food, food packaging, household products, and so on. The manufacturing of products containing or utilizing nanomaterials in their composition may also present potential toxicologic concerns in the workplace. The molecular complexity and composition of these nanomaterials are ever increasing, and the means and methods being applied to characterize and perform useful toxicologic assessments are rapidly advancing. This article includes presentations by experienced toxicologists in the nanotoxicology community who are focused on the applied aspect of the discipline toward supporting state of the art toxicologic assessments for food products and packaging, pharmaceuticals and medical devices, inhaled nanoparticle and gastrointestinal exposures, and addressing occupational safety and health issues and concerns. This symposium overview article summarizes 5 talks that were presented at the 35th Annual meeting of the American College of Toxicology on the subject of "Applied Nanotechnology." |
Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes.
Shvedova AA , Yanamala N , Kisin ER , Khailullin TO , Birch ME , Fatkhutdinova LM . PLoS One 2016 11 (3) e0150628 BACKGROUND: As the application of carbon nanotubes (CNT) in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT) could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans. METHODS: In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8), were compared with expression profiles of non-exposed (n = 7) workers (e.g., professional and/or technical staff) from the same manufacturing facility. RESULTS: Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs. CONCLUSION: This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and miRNAs as prognostic markers of MWCNT exposures in humans. |
Fibrosis biomarkers in workers exposed to MWCNTs
Fatkhutdinova LM , Khaliullin TO , Vasil'yeva OL , Zalyalov RR , Mustafin IG , Kisin ER , Birch ME , Yanamala N , Shvedova AA . Toxicol Appl Pharmacol 2016 299 125-31 Multi-walled carbon nanotubes (MWCNT) with their unique physico-chemical properties offer numerous technological advantages and are projected to drive the next generation of manufacturing growth. As MWCNT have already found utility in different industries including construction, engineering, energy production, space exploration and biomedicine, large quantities of MWCNT may reach the environment and inadvertently lead to human exposure. This necessitates the urgent assessment of their potential health effects in humans. The current study was carried out at NanotechCenter Ltd. Enterprise (Tambov, Russia) where large-scale manufacturing of MWCNT along with relatively high occupational exposure levels was reported. The goal of this small cross-sectional study was to evaluate potential biomarkers during occupational exposure to MWCNT. All air samples were collected at the workplaces from both specific areas and personal breathing zones using filter-based devices to quantitate elemental carbon and perform particle analysis by TEM. Biological fluids of nasal lavage, induced sputum and blood serum were obtained from MWCNT-exposed and non-exposed workers for assessment of inflammatory and fibrotic markers. It was found that exposure to MWCNTs caused significant increase in IL-1beta, IL6, TNF-alpha, inflammatory cytokines and KL-6, a serological biomarker for interstitial lung disease in collected sputum samples. Moreover, the level of TGF-beta1 was increased in serum obtained from young exposed workers. Overall, the results from this study revealed accumulation of inflammatory and fibrotic biomarkers in biofluids of workers manufacturing MWCNTs. Therefore, the biomarkers analyzed should be considered for the assessment of health effects of occupational exposure to MWCNT in cross-sectional epidemiological studies. |
Nanotoxicology ten years later: Lights and shadows
Shvedova A , Pietroiusti A , Kagan V . Toxicol Appl Pharmacol 2016 299 1-2 The mounting societal concerns about possible and maybe even likely adverse effects of nanomaterials are reflected in a large and growing number of publications in the field of nanotoxicology. Indeed, today's search in PubMed reveals >3700 publications on the subject denoted by (toxic+nanomaterials) - quite a growth over the last decade that began with only two dozens of them up-to 2005. |
Enzymatic oxidative biodegradation of nanoparticles: Mechanisms, significance and applications
Vlasova II , Kapralov AA , Michael ZP , Burkert SC , Shurin MR , Star A , Shvedova AA , Kagan VE . Toxicol Appl Pharmacol 2016 299 58-69 Biopersistence of carbon nanotubes, graphene oxide (GO) and several other types of carbonaceous nanomaterials is an essential determinant of their health effects. Successful biodegradation is one of the major factors defining the life span and biological responses to nanoparticles. Here, we review the role and contribution of different oxidative enzymes of inflammatory cells - myeloperoxidase, eosinophil peroxidase, lactoperoxidase, hemoglobin, and xanthine oxidase - to the reactions of nanoparticle biodegradation. We further focus on interactions of nanomaterials with hemoproteins dependent on the specific features of their physico-chemical and structural characteristics. Mechanistically, we highlight the significance of immobilized peroxidase reactive intermediates vs diffusible small molecule oxidants (hypochlorous and hypobromous acids) for the overall oxidative biodegradation process in neutrophils and eosinophils. We also accentuate the importance of peroxynitrite-driven pathways realized in macrophages via the engagement of NADPH oxidase- and NO synthase-triggered oxidative mechanisms. We consider possible involvement of oxidative machinery of other professional phagocytes such as microglial cells, myeloid-derived suppressor cells, in the context of biodegradation relevant to targeted drug delivery. We evaluate the importance of genetic factors and their manipulations for the enzymatic biodegradation in vivo. Finally, we emphasize a novel type of biodegradation realized via the activation of the "dormant" peroxidase activity of hemoproteins by the nano-surface. This is exemplified by the binding of GO to cyt c causing the unfolding and 'unmasking' of the peroxidase activity of the latter. We conclude with the strategies leading to safe by design carbonaceous nanoparticles with optimized characteristics for mechanism-based targeted delivery and regulatable life-span of drugs in circulation. |
Current understanding of interactions between nanoparticles and the immune system
Dobrovolskaia MA , Shurin M , Shvedova AA . Toxicol Appl Pharmacol 2015 299 78-89 The delivery of drugs, antigens, and imaging agents benefits from using nanotechnology-based carriers. The successful translation of nanoformulations to the clinic involves thorough assessment of their safety profiles, which, among other end-points, includes evaluation of immunotoxicity. The past decade of research focusing on nanoparticle interaction with the immune system has been fruitful in terms of understanding the basics of nanoparticle immunocompatibility, developing a bioanalytical infrastructure to screen for nanoparticle-mediated immune reactions, beginning to uncover the mechanisms of nanoparticle immunotoxicity, and utilizing current knowledge about the structure-activity relationship between nanoparticles' physicochemical properties and their effects on the immune system to guide safe drug delivery. In the present review, we focus on the most prominent pieces of the nanoparticle-immune system puzzle and discuss the achievements, disappointments, and lessons learned over the past 15 years of research on the immunotoxicity of engineered nanomaterials. |
Carbon nanotubes exposure risk assessment: from toxicology to epidemiologic studies (overview of the current problem)
Fatkhutdinova LM , Khaliullin TO , Shvedova AA . Nanotechnol Russ 2015 10 501-509 Nanoscale size and fiber like structure of carbon nanotubes (CNTs) may determine high reactivity and penetration, as well as the pathogenicity of asbestos and other mineral fibers. Despite many in vitro and in vivo studies, the absence of full-scale data on CNT effects on human health clearly point out the necessity for epidemiological studies. Currently, several projects are initiated worldwide on studying health risks associated with the inhalation of industrial CNTs, including NIOSH-promoted research (United States), the European CANTES study, and the Russian CNT-ERA project. Studies comprising several successive steps, such as CNT exposure assessment in occupational settings, toxicological evaluation, and epidemiological observations, are critical for determining material safety and use criteria. |
- Page last reviewed:Feb 1, 2024
- Page last updated:Sep 16, 2024
- Content source:
- Powered by CDC PHGKB Infrastructure