Showing papers in "Archives of Toxicology in 2013"

Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis

Abstract: Oxidative stress basically defines a condition in which prooxidant-antioxidant balance in the cell is disturbed; cellular biomolecules undergo severe oxidative damage, ultimately compromising cells viability. In recent years, a number of studies have shown that oxidative stress could cause cellular apoptosis via both the mitochondria-dependent and mitochondria-independent pathways. Since these pathways are directly related to the survival or death of various cell types in normal as well as pathophysiological situations, a clear picture of these pathways for various active molecules in their biological functions would help designing novel therapeutic strategy. This review highlights the basic mechanisms of ROS production and their sites of formation; detail mechanism of both mitochondria-dependent and mitochondria-independent pathways of apoptosis as well as their regulation by ROS. Emphasis has been given on the redox-sensitive ASK1 signalosome and its downstream JNK pathway. This review also describes the involvement of oxidative stress under various environmental toxin- and drug-induced organ pathophysiology and diabetes-mediated apoptosis. We believe that this review would provide useful information about the most recent progress in understanding the mechanism of oxidative stress-mediated regulation of apoptotic pathways. It will also help to figure out the complex cross-talks between these pathways and their modulations by oxidative stress. The literature will also shed a light on the blind alleys of this field to be explored. Finally, readers would know about the ROS-regulated and apoptosis-mediated organ pathophysiology which might help to find their probable remedies in future.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Abstract: This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review

Abstract: Nanoparticles (NPs) of copper oxide (CuO), zinc oxide (ZnO) and especially nanosilver are intentionally used to fight the undesirable growth of bacteria, fungi and algae. Release of these NPs from consumer and household products into waste streams and further into the environment may, however, pose threat to the ‘non-target’ organisms, such as natural microbes and aquatic organisms. This review summarizes the recent research on (eco)toxicity of silver (Ag), CuO and ZnO NPs. Organism-wise it focuses on key test species used for the analysis of ecotoxicological hazard. For comparison, the toxic effects of studied NPs toward mammalian cells in vitro were addressed. Altogether 317 L(E)C50 or minimal inhibitory concentrations (MIC) values were obtained for algae, crustaceans, fish, bacteria, yeast, nematodes, protozoa and mammalian cell lines. As a rule, crustaceans, algae and fish proved most sensitive to the studied NPs. The median L(E)C50 values of Ag NPs, CuO NPs and ZnO NPs (mg/L) were 0.01, 2.1 and 2.3 for crustaceans; 0.36, 2.8 and 0.08 for algae; and 1.36, 100 and 3.0 for fish, respectively. Surprisingly, the NPs were less toxic to bacteria than to aquatic organisms: the median MIC values for bacteria were 7.1, 200 and 500 mg/L for Ag, CuO and ZnO NPs, respectively. In comparison, the respective median L(E)C50 values for mammalian cells were 11.3, 25 and 43 mg/L. Thus, the toxic range of all the three metal-containing NPs to target- and non-target organisms overlaps, indicating that the leaching of biocidal NPs from consumer products should be addressed.

The essential comet assay: a comprehensive guide to measuring DNA damage and repair

Abstract: The comet assay (single cell gel electrophoresis) is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues. The assay depends on the relaxation of supercoiled DNA in agarose-embedded nucleoids (the residual bodies remaining after lysis of cells with detergent and high salt), which allows the DNA to be drawn out towards the anode under electrophoresis, forming comet-like images as seen under fluorescence microscopy. The relative amount of DNA in the comet tail indicates DNA break frequency. The assay has been modified to detect various base alterations, by including digestion of nucleoids with a lesion-specific endonuclease. We describe here recent technical developments, theoretical aspects, limitations as well as advantages of the assay, and modifications to measure cellular antioxidant status and different types of DNA repair. We briefly describe the applications of this method in genotoxicity testing, human biomonitoring, and ecogenotoxicology.

Multi-cell type human liver microtissues for hepatotoxicity testing.

Abstract: Current 2-dimensional hepatic model systems often fail to predict chemically induced hepatotoxicity due to the loss of a hepatocyte-specific phenotype in culture. For more predictive in vitro models, hepatocytes have to be maintained in a 3-dimensional environment that allows for polarization and cell–cell contacts. Preferably, the model will reflect an in vivo-like multi-cell type environment necessary for liver-like responses. Here, we report the characterization of a multi-cell type microtissue model, generated from primary human hepatocytes and liver-derived non-parenchymal cells. Liver microtissues were stable and functional for 5 weeks in culture enabling, for example, long-term toxicity testing of acetaminophen and diclofenac. In addition, Kupffer cells were responsive to inflammatory stimuli such as LPS demonstrating the possibility to detect inflammation-mediated toxicity as exemplified by the drug trovafloxacin. Herewith, we present a novel 3D liver model for routine testing in 96-well format capable of reducing the risk of unwanted toxic effects in the clinic.

Metabolism of arsenic and its toxicological relevance.

Abstract: Arsenic is a worldwide environmental pollutant and a human carcinogen. It is well recognized that the toxicity of arsenicals largely depends on the oxidoreduction states (trivalent or pentavalent) and methylation levels (monomethyl, dimethyl, and trimethyl) that are present during the process of metabolism in mammals. However, presently, the specifics of the metabolic pathway of inorganic arsenicals have yet to be confirmed. In mammals, there are two possible mechanisms that have been proposed for the metabolic pathway of inorganic arsenicals, oxidative methylation, and glutathione conjugation. Oxidative methylation, which was originally proposed in fungi, is based on findings that arsenite (iAsIII) is sequentially converted to monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) in both humans and in laboratory animals such as mice and rats. However, recent in vitro observations have demonstrated that arsenic is only methylated in the presence of glutathione (GSH) or other thiol compounds, which strongly suggests that arsenic is methylated in trivalent forms. The glutathione conjugation mechanism is supported by findings that have shown that most intracellular arsenicals are trivalent and excreted from cells as GSH conjugates. Since non-conjugated trivalent arsenicals are highly reactive with thiol compounds and are easily converted to less toxic corresponding pentavalent arsenicals, the arsenic–glutathione conjugate stability may be the most important factor for determining the toxicity of arsenicals. In addition, “being a non-anionic form” also appears to be a determinant of the toxicity of oxo-arsenicals or thioarsenicals. The present review discusses both the metabolism of arsenic and the toxicity of arsenic metabolites.

Heat shock proteins and heat shock factor 1 in carcinogenesis and tumor development: an update

Abstract: Heat shock proteins (HSP) are a subset of the molecular chaperones, best known for their rapid and abundant induction by stress. HSP genes are activated at the transcriptional level by heat shock transcription factor 1 (HSF1). During the progression of many types of cancer, this heat shock transcriptional regulon becomes co-opted by mechanisms that are currently unclear, although evidently triggered in the emerging tumor cell. Concerted activation of HSF1 and the accumulation of HSPs then participate in many of the traits that permit the malignant phenotype. Thus, cancers of many histologies exhibit activated HSF1 and increased HSP levels that may help to deter tumor suppression and evade therapy in the clinic. We review here the extensive work that has been carried out and is still in progress aimed at (1) understanding the oncogenic mechanisms by which HSP genes are switched on, (2) determining the roles of HSF1/HSP in malignant transformation and (3) discovering approaches to therapy based on disrupting the influence of the HSF1-controlled transcriptome in cancer.

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach

Abstract: Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)- derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (\20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles. © The Author(s) 2012.

Cytotoxicity of hydroxyapatite nanoparticles is shape and cell dependent.

Abstract: Nanosized hydroxyapatite (nHA) has been proposed as drug delivery vehicles because of its biocompatibility. While the possible risks of nHA inducing inflammation have been highlighted, the specific influence of varying nHA particle morphology is still unclear. In order to establish this understanding, nHA of four different shapes—needle (nHA-ND), plate (nHA-PL), sphere (nHA-SP) and rod (nHA-RD)—were synthesized. The particle effects with the concentration of 10–300 μg/mL on cytotoxicity, oxygen species generation, production of inflammatory cytokines (TNF-α and IL-6), particle–cell association and cellular uptake were evaluated on BEAS-2B and RAW264.7 cells. Results show that nHA-ND and nHA-PL induced the most significant cell death in BEAS-2B cultures compared to nHA-SP and nHA-RD. Necrosis–apoptosis assay by FITC Annexin V and propidium iodide (PI) staining revealed loss of the majority of BEAS-2B by necrosis. No significant cell death was recorded in RAW264.7 cultures exposed to any of the nHA groups. Correspondingly, no significant differences were observed in TNF-α level for RAW264.7 cells upon incubation with nHA of different shapes. In addition, nHA-RD exhibited a higher degree of particle–cell association and internalization in both BEAS-2B and RAW264.7 cells, compared to nHA-ND. The phenomena suggested that higher particle–cell association and increased cellular uptake of nHA need not result in increased cytotoxicity, indicating the importance of particle shape on cytotoxicity. Specifically, needle- and plate-shaped nHA induced the most significant cell-specific cytotoxicity and IL-6 expression but showed the least particle–cell association. Taken collectively, we demonstrated the shape-dependent effects of nHA on cytotoxicity, inflammatory cytokine expression and particle–cell association.

Cadmium and cellular signaling cascades: interactions between cell death and survival pathways.

Abstract: Cellular stress elicited by the toxic metal Cd2+ does not coerce the cell into committing to die from the onset. Rather, detoxification and adaptive processes are triggered concurrently, allowing survival until normal function is restored. With high Cd2+, death pathways predominate. However, if sublethal stress levels affect cells for prolonged periods, as in chronic low Cd2+ exposure, adaptive and survival mechanisms may deregulate, such that tumorigenesis ensues. Hence, death and malignancy are the two ends of a continuum of cellular responses to Cd2+, determined by magnitude and duration of Cd2+ stress. Signaling cascades are the key factors affecting cellular reactions to Cd2+. This review critically surveys recent literature to outline major features of death and survival signaling pathways as well as their activation, interactions and cross talk in cells exposed to Cd2+. Under physiological conditions, receptor activation generates 2nd messengers, which are short-lived and act specifically on effectors through their spatial and temporal dynamics to transiently alter effector activity. Cd2+ recruits physiological 2nd messenger systems, in particular Ca2+ and reactive oxygen species (ROS), which control key Ca2+- and redox-sensitive molecular switches dictating cell function and fate. Severe ROS/Ca2+ signals activate cell death effectors (ceramides, ASK1-JNK/p38, calpains, caspases) and/or cause irreversible damage to vital organelles, such as mitochondria and endoplasmic reticulum (ER), whereas low localized ROS/Ca2+ levels act as 2nd messengers promoting cellular adaptation and survival through signal transduction (ERK1/2, PI3K/Akt-PKB) and transcriptional regulators (Ref1-Nrf2, NF-κB, Wnt, AP-1, bestrophin-3). Other cellular proteins and processes targeted by ROS/Ca2+ (metallothioneins, Bcl-2 proteins, ubiquitin–proteasome system, ER stress-associated unfolded protein response, autophagy, cell cycle) can evoke death or survival. Hence, temporary or permanent disruptions of ROS/Ca2+ induced by Cd2+ play a crucial role in eliciting, modulating and linking downstream cell death and adaptive and survival signaling cascades.

Cellular and molecular mechanisms of hepatocellular carcinoma: an update.

Abstract: Hepatocellular carcinoma (HCC) is the most common primary malignant tumor that accounts for ~80 % of all liver cancer cases worldwide. It is a multifactorial disease caused by a variety of risk factors and often develops in the background of underlying cirrhosis. A number of cellular phenomena, such as tumor microenvironment, inflammation, oxidative stress, and hypoxia act in concert with various molecular events to facilitate tumor initiation, progression, and metastasis. The emergence of microRNAs and molecular-targeted therapies adds a new dimension in our efforts to combat this deadly disease. Intense research in this multitude of areas has led to significant progress in our understanding of cellular processes and molecular mechanisms that occur during multistage events that lead to hepatocarcinogenesis. In this review, we discuss the current knowledge of HCC, focusing mainly on advances that have occurred during the past 5 years and on the development of novel therapeutics for liver cancer.

Inflammatory findings on species extrapolations: humans are definitely no 70-kg mice

Abstract: Modern toxicology has embraced in vitro methods, and major hopes are based on the Omics technologies and systems biology approaches they bring along (Hartung and McBride in ALTEX 28(2):83-93, 2011; Hartung et al. in ALTEX 29(2):119-28, 2012). A culture of stringent validation has been developed for such approaches (Leist et al. in ALTEX 27(4):309-317, 2010; ALTEX 29(4):373-88, 2012a; Toxicol Res 1:8-22, 2012b), while the quality and usefulness of animal experiments have been little scrutinized. A new study (Seok et al. 2013) now shows the low predictivity of animal responses in the field of inflammation. These findings corroborate earlier findings from comparisons in the fields of neurodegeneration, stroke and sepsis. The low predictivity of animal experiments in research areas allowing direct comparisons of mouse versus human data puts strong doubt on the usefulness of animal data as key technology to predict human safety.

Protection by chrysin, apigenin, and luteolin against oxidative stress is mediated by the Nrf2-dependent up-regulation of heme oxygenase 1 and glutamate cysteine ligase in rat primary hepatocytes

Abstract: Chrysin, apigenin, and luteolin are flavones that differ in their number of hydroxyl groups in the B ring. In this study, we investigated the protection by chrysin, apigenin, and luteolin against tert-butyl hydroperoxide (tBHP)-induced oxidative stress and the possible mechanisms involved in rat primary hepatocytes. Chrysin, apigenin, and luteolin dose-dependently up-regulated the protein expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase (GCL) catalytic (GCLC) and modifier subunit (GCLM) and increased the intracellular glutathione (GSH) content and the ratio of GSH to oxidized GSH. Among the flavones studied, chrysin showed the greatest induction of HO-1, GCLC, and GCLM protein expression and GSH content. Cellular reactive oxygen species production induced by tBHP was attenuated by pretreatment with chrysin, apigenin, and luteolin (P < .05), and this protection was reversed by the GCL inhibitor l-buthionine-S-sulfoximine and the HO-1 inhibitor zinc protoporphyrin. Chrysin, apigenin, and luteolin activated extracellular signal-regulated protein kinase 2 (ERK2), nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, nuclear Nrf2–antioxidant responsive element (ARE) binding activity, and ARE-dependent luciferase activity. Both ERK2 and Nrf2 siRNAs attenuated chrysin-induced HO-1, GCLC, and GCLM protein expression. Taken together, these results suggest that chrysin, apigenin, and luteolin inhibit tBHP-induced oxidative stress by up-regulating HO-1, GCLC, and GCLM gene transcription via the ERK2/Nrf2/ARE signaling pathways in rat primary hepatocytes.

Relative oral bioavailability of 3-MCPD from 3-MCPD fatty acid esters in rats

Abstract: In order to quantify the relative oral bioavailability of 3-chloropropane-1,2-diol (3-MCPD) from 3-MCPD fatty acid diesters in vivo, 1,2-dipalmitoyl-3-chloropropane-1,2-diol (3-MCPD diester) and 3-MCPD were orally applied to rats in equimolar doses. In both cases, the time courses of 3-MCPD concentrations were measured in blood, various organs, tissues and intestinal luminal contents. The results show that 3-MCPD is released by enzymatic hydrolysis from the 3-MCPD diester in the gastrointestinal tract and distributed to blood, organs and tissues. Based on the measurements in blood, the areas under the curve (AUC) for 3-MCPD were calculated. By comparing both AUC, the relative amount of 3-MCPD bioavailable from the 3-MCPD diester was calculated to be 86 % on average of the amount bioavailable following administration of 3-MCPD. In view of limited experimental data, it is justified for the purpose of risk assessment to assume complete hydrolysis of the diesters in the gastro-intestinal tract. Therefore, assessment of the extent of exposure to 3-MCPD released from its fatty acid esters should be performed in the same way as exposure to the same molar quantity of 3-MCPD.

Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.

Abstract: Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants.

An overview of transcriptional regulation in response to toxicological insult

Abstract: The completion of the human genome project and the subsequent advent of DNA microarray and high-throughput sequencing technologies have led to a renaissance in molecular toxicology. Toxicogenomic data sets, from both in vivo and in vitro studies, are growing exponentially, providing a wealth of information on regulation of stress pathways at the transcriptome level. Through such studies, we are now beginning to appreciate the diversity and complexity of biological responses to xenobiotics. In this review, we aim to consolidate and summarise the major toxicologically relevant transcription factor-governed molecular pathways. It is becoming clear that different chemical entities can cause oxidative, genotoxic and proteotoxic stress, which induce cellular responses in an effort to restore homoeostasis. Primary among the response pathways involved are NFE2L2 (Nrf2), NFE2L1 (Nrf1), p53, heat shock factor and the unfolded protein response. Additionally, more specific mechanisms exist where xenobiotics act as ligands, including the aryl hydrocarbon receptor, metal-responsive transcription factor-1 and the nuclear receptor family of transcription factors. Other pathways including the immunomodulatory transcription factors NF-κB and STAT together with the hypoxia-inducible transcription factor HIF are also implicated in cellular responses to xenobiotic exposure. A less specific but equally important aspect to cellular injury controlled by transcriptional activity is loss of tissue-specific gene expression, resulting in dedifferentiation of target cells and compromise of tissue function. Here, we review these pathways and the genes they regulate in order to provide an overview of this growing field of molecular toxicology.

Genotoxic and carcinogenic potential of engineered nanoparticles: an update

Abstract: Nanoscience and nanotechnology have seen an exponential growth over the past decade largely due to the unique properties of engineered nanoparticles (ENPs), advances in ENP synthesis, and imaging or analysis tools. The unique properties such as high surface area to volume ratio, abundant reactive sites on the surface, large fraction of atoms located on the exterior face have made these novel materials the most sought after for consumer and industrial applications. This significant increase in the ENP containing consumer products has also enhanced the chances of human and environmental exposure. Humans get exposed to ENPs at various steps of its synthesis (laboratory), manufacture (industry), use (consumer products, devices, medicines, etc.) and through the environment (contaminated water, aerosolized particles, and disposal). Such exposures to ENPs are known to induce genotoxicity, cytotoxicity, and carcinogenicity in biological system. This is attributed to several factors, such as direct interaction of ENPs with the genetic material, indirect damage due to reactive oxygen species generation, release of toxic ions from soluble ENPs, interaction with cytoplasmic/nuclear proteins, binding with mitotic spindle or its components, increased oxidative stress, disturbance of cell cycle checkpoint functions, inhibition of antioxidant defense, and many others. The present review describes an overview of in vitro and in vivo genotoxicity studies with ENPs, advantages and potential problems associated with the methods used in genotoxicity assessment, and the need for appropriate method and approach for risk assessment of ENPs.

Nanosilver: application and novel aspects of toxicology

Abstract: Nanomaterials are a challenge to toxicology. The high diversity of novel materials and products will require extensive expertize for evaluation and regulatory efforts. Nanomaterials are of substantial scientific and economic potential. Here, we will focus on nanosilver, a material not only with medical applications, but a rapidly increasing use in surprisingly many products. Consequently, toxicological evaluation has to cover an increasing range of complex topics. The toxicology of nanosilver is advancing rapidly; regulatory efforts by Federal Drug Agency and European Environment Protection Agencies are substantial. Current toxicological data, ranging from in vitro studies with cell lines to rodent experiments and ecological evaluation, are numerous, and many groups are providing continuously new data. However, standard classification based on nanosize only is neglecting nanoshape, which adds another level of complexity to the analysis of biological effects. A surprising neglect in nanosilver toxicology so far is the analysis of effects of nanosilver on amyloidosis. Amyloid diseases are widespread in humans and a severe health hazard. The known potential of silver to stimulate amyloidosis in rodents will require a timely and balanced evaluation of nanosilvers.

Size influences the cytotoxicity of poly (lactic-co-glycolic acid) (PLGA) and titanium dioxide (TiO 2 ) nanoparticles

Abstract: The aim of this study is to uncover the size influence of poly (lactic-co-glycolic acid) (PLGA) and titanium dioxide (TiO2) nanoparticles on their potential cytotoxicity. PLGA and TiO2 nanoparticles of three different sizes were thoroughly characterized before in vitro cytotoxic tests which included viability, generation of reactive oxygen species (ROS), mitochondrial depolarization, integrity of plasma membrane, intracellular calcium influx and cytokine release. Size-dependent cytotoxic effect was observed in both RAW264.7 cells and BEAS-2B cells after cells were incubated with PLGA or TiO2 nanoparticles for 24 h. Although PLGA nanoparticles did not trigger significantly lethal toxicity up to a concentration of 300 μg/ml, the TNF-α release after the stimulation of PLGA nanoparticles should not be ignored especially in clinical applications. Relatively more toxic TiO2 nanoparticles triggered cell death, ROS generation, mitochondrial depolarization, plasma membrane damage, intracellular calcium concentration increase and size-dependent TNF-α release, especially at a concentration higher than 100 μg/ml. These cytotoxic effects could be due to the size-dependent interaction between nanoparticles and biomolecules, as smaller particles tend to adsorb more biomolecules. In summary, we demonstrated that the ability of protein adsorption could be an important paradigm to predict the in vitro cytotoxicity of nanoparticles, especially for low toxic nanomaterials such as PLGA and TiO2 nanoparticles.

MicroRNA-210 targets antiapoptotic Bcl-2 expression and mediates hypoxia-induced apoptosis of neuroblastoma cells

Abstract: MicroRNAs (miRNAs) can regulate cell survival and death by targeting apoptosis-related gene expression. miR-210 is one of the most hypoxia-sensitive miRNAs. In this study, we evaluated the roles of miR-210 in hypoxia-induced insults to neural cells. Treatment of neuro-2a cells with oxygen/glucose deprivation (OGD) induced cell apoptosis in a time-dependent manner. In parallel, OGD time-dependently increased cellular miR-210 levels. Knocking down miR-210 expression using specific antisenses significantly attenuated OGD-induced neural apoptosis. Concurrently, OGD increased hypoxia-inducible factor (HIF)-1α mRNA and protein syntheses. Pretreatment with YC-1, an inhibitor of HIF-1α, reduced OGD-caused cell death. Sequentially, OGD specifically decreased antiapoptotic Bcl-2 mRNA and protein levels in neuro-2a cells. A search by a bioinformatic approach revealed that miR-210-specific binding elements exist in the 3′-untranslated region of Bcl-2 mRNA. Application of miR-210 antisenses simultaneously alleviated OGD-involved inhibition of Bcl-2 mRNA expression. In comparison, overexpression of miR-210 synergistically diminished OGD-caused inhibition of Bcl-2 mRNA expression and consequently induced greater cellular insults. Taken together, this study shows that OGD can induce miR-210 expression through activating HIF-1α. And miR-210 can mediate hypoxia-induced neural apoptosis by targeting Bcl-2.

Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies

Abstract: Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed

Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity

Abstract: Primary human hepatocytes (PHH) are the “gold standard” for in vitro toxicity tests. However, 2D PHH cultures have limitations that are due to a time-dependent dedifferentiation process visible by morphological changes closely connected to a decline of albumin production and CYP450 activity. The 3D in vitro culture corresponds to in vivo-like tissue architecture, which preserves functional characteristics of hepatocytes, and therefore can at least partially overcome the restrictions of 2D cultures. Consequently, several drug toxicities observed in vivo cannot be reproduced in 2D in vitro models, for example, the toxic effects of acetaminophen. The objective of this study was to identify molecular differences between 2D and 3D cultivation which explain the observed toxicity response. Our data demonstrated an increase in cell death after treatment with acetaminophen in 3D, but not in 2D cultures. Additionally, an acetaminophen concentration-dependent increase in the CYP2E1 expression level in 3D cultures was detected. However, during the treatment with 10 mM acetaminophen, the expression level of SOD gradually decreased in 3D cultures and was undetectable after 24 h. In line with these findings, we observed higher import/export rates in the membrane transport protein, multidrug resistance-associated protein-1, which is known to be specific for acetaminophen transport. The presented data demonstrate that PHH cultured in 3D preserve certain metabolic functions. Therefore, they have closer resemblance to the in vivo situation than PHH in 2D cultures. In consequence, 3D cultures will allow for a more accurate hepatotoxicity prediction in in vitro models in the future.

Recent trend in risk assessment of formaldehyde exposures from indoor air

Abstract: Studies about formaldehyde (FA) published since the guideline of 0.1 mg/m3 by the World Health Organization (WHO) in 2010 have been evaluated; critical effects were eye and nasal (portal-of-entry) irritation. Also, it was considered to prevent long-term effects, including all types of cancer. The majority of the recent toxicokinetic studies showed no exposure-dependent FA–DNA adducts outside the portal-of-entry area and FA–DNA adducts at distant sites were due to endogenously generated FA. The no-observed-adverse-effect level for sensory irritation was 0.5 ppm and recently reconfirmed in hypo- and hypersensitive individuals. Investigation of the relationship between FA exposure and asthma or other airway effects in children showed no convincing association. In rats, repeated exposures showed no point mutation in the p53 and K-Ras genes at ≤15 ppm neither increased cell proliferation, histopathological changes and changes in gene expression at 0.7 ppm. Repeated controlled exposures (0.5 ppm with peaks at 1 ppm) did not increase micronucleus formation in human buccal cells or nasal tissue (0.7 ppm) or in vivo genotoxicity in peripheral blood lymphocytes (0.7 ppm), but higher occupational exposures were associated with genotoxicity in buccal cells and cultivated peripheral blood lymphocytes. It is still valid that exposures not inducing nasal squamous cell carcinoma in rats will not induce nasopharyngeal cancer or lymphohematopoietic malignancies in humans. Reproductive and developmental toxicity are not considered relevant in the absence of sensory irritation. In conclusion, the WHO guideline has been strengthened.

Dioscin-induced autophagy mitigates cell apoptosis through modulation of PI3K/Akt and ERK and JNK signaling pathways in human lung cancer cell lines.

Abstract: Our previous study has revealed that dioscin, a compound with anti-inflammatory, lipid-lowering, anticancer and hepatoprotective effects, may induce autophagy in hepatoma cells. Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. In this study, the role of autophagy and related signaling pathways during dioscin-induced apoptosis in human lung cancer cells was investigated. Results from 4′-6-diamidino-2-phenylindole and annexin-V/PI double-staining assay showed that caspase-3- and caspase-8-dependent, and dose-dependent apoptoses were detected after a 24-h dioscin treatment. Meanwhile, autophagy was detected as early as 12 h after an exposure to low-dose dioscin, as indicated by an up-regulated expression of LC3-II and beclin-1 proteins. Blockade of autophagy with bafilomycin A1 or 3-methyladenine sensitized the A549 and H1299 cells to apoptosis. Treatment of A549 and H1299 cells with dioscin caused a dose-dependent increase in ERK1/2 and JNK1/2 activity, accompanied with a decreased PI3K expression and decreased phosphorylation of Akt and mTOR. Taken together, this study demonstrated for the first time that autophagy occurred earlier than apoptosis during dioscin-induced human lung cancer cell line apoptosis. Dioscin-induced autophagy via ERK1/2 and JNK1/2 pathways may provide a protective mechanism for cell survival against dioscin-induced apoptosis to act as a cytoprotective reaction.

Neutralization of interleukin-1 beta attenuates silica-induced lung inflammation and fibrosis in C57BL/6 mice

Abstract: The inflammation and fibrosis induced by silica dust are considered to be substantial responses in silicosis progression Interleukin-1 beta (IL-1β) plays an important role in silica-induced lung inflammation, but the mechanisms that underlie the influence of IL-1β on the progression of silicosis remain unclear In this study, the role of IL-1β in silica-induced inflammation and fibrosis was evaluated by administering a suspension of 25-mg silica dust, either with or without 40 μg anti-mouse IL-1β monoclonal antibody (mAb), to the lungs of male C57BL/6 mice Silica + anti-IL-1β mAb-treated mice showed the depletion of IL-1β as well as the attenuation of inflammation, as evaluated in the bronchoalveolar lavage fluid (BALF) and histological sections from 1 to 84 days after silica exposure Further study of the BALF indicated that inhibition of IL-1β could reduce the contents of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 The real-time PCR and pathology results showed that the neutralization of IL-1β attenuated silica-induced fibrosis by inhibiting the gene expression of transforming growth factor-beta 1, collagen I and fibronectin The examination of Th1-cytokine and Th2-cytokine suggested that depletion of IL-1β decelerated the Th1/Th2 balance toward a Th2-dominant response In conclusion, the present study suggests that the neutralization of IL-1β attenuates silica-induced inflammation and fibrosis by inhibiting other inflammatory and fibrogenic mediators and modulating the Th1/Th2 balance

Metabolism of the plasticizer and phthalate substitute diisononyl-cyclohexane-1,2-dicarboxylate (DINCH(®)) in humans after single oral doses.

Abstract: Hexamoll® DINCH® (diisononyl-cyclohexane-1,2-dicarboxylate) is a new high-molecular-weight plasticizer and a phthalate substitute. In this study, the metabolism of DINCH® was investigated by oral dosage of three male volunteers with approximately 50 mg Hexamoll® DINCH® (resulting in individual doses between 0.552 and 0.606 mg/kg body weight). Their urine samples were consecutively collected over 48 h. In analogy to di-iso-nonylphthalate (DINP) metabolism, we quantified the simple monoester mono-isononyl-cyclohexane-1,2-dicarboxylate (MINCH) and its secondary oxidized metabolites with HPLC–MS/MS via isotope dilution analysis. Additionally, we quantified the unspecific full breakdown product, cyclohexane-1,2-dicarboxylic acid (CHDA), via standard addition. All postulated metabolites were present in all samples analyzed. The unspecific CHDA was identified as the major urinary metabolite representing 23.7 % of the dose as the mean of the three volunteers (range 20.0–26.5 %). 14.8 % (11.3–16.7 %) of the dose was excreted as monoesters with oxidative modifications, in particular OH-MINCH 10.7 % (7.7–12.9 %), oxo-MINCH 2.0 % (1.5–2.6 %) and carboxy-MINCH 2.0 % (1.8–2.3 %). Less than 1 % was excreted as the simple monoester MINCH. In sum, 39.2 % (35.9–42.4 %) of the DINCH® dose was excreted as these metabolites in urine within 48 h. Over 90 % of the metabolites investigated were excreted within 24 h after application. The secondary oxidized metabolites, with elimination half-times between 10 and 18 h, proved to be apt and specific biomarkers to determine DINCH® exposure. With this study, we provide reliable urinary excretion factors to calculate DINCH® intakes based on these metabolites in environmental and occupational studies.

Comparison of wood smoke PM2.5 obtained from the combustion of FIR and beech pellets on inflammation and DNA damage in A549 and THP-1 human cell lines.

Abstract: The aim of this study was to investigate the effect on the induction of interleukin-8 of particulate matter (PM) from fir and beech pellets burnt in domestic appliances on two human cells lines, namely the lung epithelial cell line A549 and the promyelocytic cell line THP-1. The effects of PM2.5 obtained from combustion of beech and fir pellets were compared to reference diesel exhaust particulates (DEP). In parallel, wood smoke PM-induced genotoxicity and oxidative stress were also investigated in A549 cells. Cells were treated for different times (3–72 h) with increasing concentrations of PM2.5 obtained from sequential combustions of fir and beech pellets or reference DEP. Cell viability was assessed by lactate dehydrogenase leakage, and the release of interleukin-8 or CXCL8 (IL-8) was measured to evaluate the pro-inflammatory effect. Oxidative stress was evaluated by the 5(6)-carboxy-2′,7′dichlorofluorescein diacetate (DCFH-DA) assay and DNA damage by the alkaline comet assay and micronucleus frequency by flow cytometry. Both A549 and THP-1 cells responded in a dose- and time-related manner to wood smoke PM2.5 with IL-8 release, particles obtained from late combustions being the most active. THP-1 cells were more sensitive than A549 cells. On a mass base, similar effects were observed for both fir and beech PM2.5. However, the combustion of beech pellets generated approximately three times more PM2.5 than fir pellets. Regarding the mechanism of PM2.5 uptake, in both THP-1 and A549 cells, cytochalasin D prevented PM2.5-induced IL-8 mRNA expression and cytokine release, indicating a key role for actin polymerization in particles uptake and that the production of IL-8 correlated with particle phagocytosis. As signal transduction pathway involvement, in both THP-1 and A549 cells, PM2.5-induced IL-8 release could be completely blocked by the selective inhibitor SB203580, indicating a role of p38 MAPK activation. PM2.5 from both fir and beech pellets also induced modest DNA lesions dose related, measured as strand breaks, whereas no increase in the number of micronucleus was observed. Similar effects were observed with DEP, arguing against less dangerous effects of wood smoke particles than other categories of combustion-derived particles in the same size range. Overall, results suggest that combustion conditions can significantly affect the characteristics of particles and the consequent toxicity, and that different woods can generate different amounts of PM2.5.

Mammalian toxicology and human exposures to the flame retardant 2,2′,6,6′-tetrabromo-4,4′-isopropylidenediphenol (TBBPA): implications for risk assessment

Abstract: The compound 2,2′,6,6′-Tetrabromo-4,4′-isopropylidenediphenol (tetrabromobisphenol A, TBBPA) is used as a reactive and additive flame retardant. This review evaluates the mammalian toxicology of TBBPA and summarizes recent human exposure and risk assessments. TBBPA has a low potential for systemic or reproductive toxicity, and no-observed-adverse-effect-levels were greater than 1,000 mg/kg body weight (bw)/day in a 90-day oral toxicity study, a developmental toxicity study and a two-generation reproductive and developmental toxicity study. Some interactions of TBBPA with hormone-mediated pathways were noted in vitro; however, when studied in vivo, TBBPA did not produce adverse effects that might be considered to be related to disturbances in the endocrine system. Therefore, in accordance with internationally accepted definitions, TBBPA should not be considered an “endocrine disruptor.” Furthermore, TBBPA is rapidly excreted in mammals and therefore does not have a potential for bioaccumulation. Measured concentrations of TBBPA in house dust, human diet and human serum samples are very low. Daily intakes of TBBPA in humans were estimated to not exceed a few ng/kg bw/day. Due to the low exposures and the low potential for toxicity, margins of exposures for TBBPA in the human population were between 6 × 104 (infants) to 6 × 107 (adults). Exposures of the general population are also well below the derived-no-effect-levels derived for endpoints of potential concern in REACH.

Size of TiO 2 nanoparticles influences their phototoxicity: an in vitro investigation

Abstract: To uncover the size influence of TiO2 nanoparticles on their potential toxicity, the cytotoxicity of different-sized TiO2 nanoparticles with and without photoactivation was tested. It was demonstrated that without photoactivation, TiO2 nanoparticles were inert up to 100 μg/ml. On the contrary, with photoactivation, the toxicity of TiO2 nanoparticles significantly increased, which correlated well with the specific surface area of the particles. Our results also suggest that the generation of hydroxyl radicals and reactive oxygen species (ROS)-mediated damage to the surface-adsorbed biomolecules could be the two major reasons for the cytotoxicity of TiO2 nanoparticles after photoactivation. Higher ROS generation from smaller particles was detected under both biotic and abiotic conditions. Smaller particles could adsorb more proteins, which was confirmed by thermogravimetric analysis. To further investigate the influence of the generation of hydroxyl radicals and adsorption of protein, poly (ethylene-alt-maleic anhydride) (PEMA) and chitosan were used to coat TiO2 nanoparticles. The results confirmed that surface coating of TiO2 nanoparticles could reduce such toxicity after photoactivation, by hindering adsorption of biomolecules and generation of hydroxyl radical (·OH) during photoactivation.

Origin of the linearity no threshold (LNT) dose–response concept

Abstract: This paper identifies the origin of the linearity at low-dose concept [i.e., linear no threshold (LNT)] for ionizing radiation-induced mutation. After the discovery of X-ray-induced mutations, Olson and Lewis (Nature 121(3052):673-674, 1928) proposed that cosmic/terrestrial radiation-induced mutations provide the principal mechanism for the induction of heritable traits, providing the driving force for evolution. For this concept to be general, a LNT dose relationship was assumed, with genetic damage proportional to the energy absorbed. Subsequent studies suggested a linear dose response for ionizing radiation-induced mutations (Hanson and Heys in Am Nat 63(686):201-213, 1929; Oliver in Science 71:44-46, 1930), supporting the evolutionary hypothesis. Based on an evaluation of spontaneous and ionizing radiation-induced mutation with Drosophila, Muller argued that background radiation had a negligible impact on spontaneous mutation, discrediting the ionizing radiation-based evolutionary hypothesis. Nonetheless, an expanded set of mutation dose-response observations provided a basis for collaboration between theoretical physicists (Max Delbruck and Gunter Zimmer) and the radiation geneticist Nicolai Timofeeff-Ressovsky. They developed interrelated physical science-based genetics perspectives including a biophysical model of the gene, a radiation-induced gene mutation target theory and the single-hit hypothesis of radiation-induced mutation, which, when integrated, provided the theoretical mechanism and mathematical basis for the LNT model. The LNT concept became accepted by radiation geneticists and recommended by national/international advisory committees for risk assessment of ionizing radiation-induced mutational damage/cancer from the mid-1950s to the present. The LNT concept was later generalized to chemical carcinogen risk assessment and used by public health and regulatory agencies worldwide.