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To improve the mechanistic screening of reproductive toxicants in chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood–testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases. [ABSTRACT FROM AUTHOR]

New approach methodologies (NAM), including omics and in vitro approaches, are contributing to the implementation of 3R (reduction, refinement and replacement) strategies in regulatory science and risk assessment. In this study, we present an integrative transcriptomics and proteomics analysis workflow for the validation and revision of complex fish genomes and demonstrate how proteogenomics expression matrices can be used to support multi-level omics data integration in non-model species in vivo and in vitro. Using Atlantic salmon as an example, we constructed proteogenomic databases from publicly available transcriptomic data and in-house generated RNA-Seq and LC-MS/MS data. Our analysis identified ∼80,000 peptides, providing direct evidence of translation for over 40,000 RefSeq structures. The data also highlighted 183 co-located peptide groups that supported a single transcript each, and in each case, either corrected a previous annotation, supported Ensembl annotations not present in RefSeq, or identified novel previously unannotated genes. Proteogenomics data-derived expression matrices revealed distinct profiles for the different tissue types analyzed. Focusing on proteins involved in defense against xenobiotics, we detected distinct expression patterns across different salmon tissues and observed homology in the expression of chemical defense proteins between in vivo and in vitro liver systems. Our study demonstrates the potential of proteogenomic analyses in extending our understanding of complex fish genomes and provides an advanced bioinformatic toolkit to support the further development of NAMs and their application in regulatory science and (eco)toxicological studies of non-model species.

Chemical risk assessment ensures protection from the toxic effects of drugs and manmade chemicals. To comply with regulatory guidance, studies in complex organisms are required, as well as mechanistic studies to establish the relevance of any toxicities observed to man. Although in vitro toxicity models are improving, in vivo studies remain central to this process. Such studies are invariably time-consuming and often involve large numbers of animals. New regulatory frameworks recommend the implementation of “smart” in vivo approaches to toxicity testing that can effectively assess safety for humans and comply with societal expectations for reduction in animal use. A major obstacle in reducing the animals required is the time-consuming and complexity of the pathological endpoints used as markers of toxicity. Such endpoints are prone to inter-animal variability, subjectivity and require harmonisation between testing sites. As a consequence, large numbers of animals per experimental group are required. To address this issue, we propose the implementation of sophisticated stress response reporter mice that we have developed. These reporter models provide early biomarkers of toxic potential in a highly reproducible manner at single-cell resolution, which can also be measured non-invasively and have been extensively validated in academic research as early biomarkers of stress responses for a wide range of chemicals at human-relevant exposures. In this report, we describe a new and previously generated models in our lab, provide the methodology required for their use and discuss how they have been used to inform on toxic risk (likelihood of chemical causing an adverse health effect). We propose our in vivo approach is more informative (refinement) and reduces the animal use (reduction) compared to traditional toxicity testing. These models could be incorporated into tiered toxicity testing and used in combination with in vitro assays to generate quantitative adverse outcome pathways and inform on toxic potential.

We present a mechanistic machine-learning quantitative structure-activity relationship (QSAR) model to predict mammalian acute oral toxicity. We trained our model using a rat acute toxicity database compiled by the US National Toxicology Program. We profiled the database using new and published profilers and identified the most plausible mechanisms that drive high acute toxicity (LD50 ≤ 50 mg/kg; GHS categories 1 or 2). Our QSAR model assigns primary mechanisms to compounds, followed by predicting their acute oral LD50 using a random-forest machine-learning model. These predictions were further refined based on structural and mechanistic read-across to substances within the training set. Our model is optimized for sensitivity and aims to minimize the likelihood of underpredicting the toxicity of assessed compounds. It displays high sensitivity (76.1% or 76.6% for compounds in GHS 1–2 or GHS 1–3 categories, respectively), coupled with ≥73.7% balanced accuracy. We further demonstrate the utility of undertaking a mechanistic approach when predicting the toxicity of compounds acting via a rare mode of action (MOA) (aconitase inhibition). The mechanistic profilers and framework of our QSAR model are route- and toxicity endpoint-agnostic, allowing for future applications to other endpoints and routes of administration. Furthermore, we present a preliminary exploration of the potential role of metabolic clearance in acute toxicity. To the best of our knowledge, this effort represents the first accurate mechanistic QSAR model for acute oral toxicity that combines machine learning with MOA assignment, while also seeking to minimize underprediction of more highly potent substances. [ABSTRACT FROM AUTHOR]

Chemical risk assessment ensures protection from the toxic effects of drugs and manmade chemicals. To comply with regulatory guidance, studies in complex organisms are required, as well as mechanistic studies to establish the relevance of any toxicities observed to man. Although in vitro toxicity models are improving, in vivo studies remain central to this process. Such studies are invariably time-consuming and often involve large numbers of animals. New regulatory frameworks recommend the implementation of “smart” in vivo approaches to toxicity testing that can effectively assess safety for humans and comply with societal expectations for reduction in animal use. A major obstacle in reducing the animals required is the time-consuming and complexity of the pathological endpoints used as markers of toxicity. Such endpoints are prone to inter-animal variability, subjectivity and require harmonisation between testing sites. As a consequence, large numbers of animals per experimental group are required. To address this issue, we propose the implementation of sophisticated stress response reporter mice that we have developed. These reporter models provide early biomarkers of toxic potential in a highly reproducible manner at single-cell resolution, which can also be measured non-invasively and have been extensively validated in academic research as early biomarkers of stress responses for a wide range of chemicals at human-relevant exposures. In this report, we describe a new and previously generated models in our lab, provide the methodology required for their use and discuss how they have been used to inform on toxic risk. We propose our in vivo approach is more informative (refinement) and reduces the animal use (reduction) compared to traditional toxicity testing. These models could be incorporated into tiered toxicity testing and used in combination with in vitro assays to generate quantitative adverse outcome pathways and inform on toxic potential.

Nanomaterials (NMs) can be produced in plenty of variants posing several challenges for NM hazard and risk assessment. Metabolomic profiling of NM-treated cells and tissues allows for insights into underlying Mode-of-Action (MoA) and offers several advantages in this context. It supports the description of Adverse Outcome Pathways (AOPs) and, therefore, tailored AOP-based hazard testing strategies. Moreover, it bears great potential for biomarker discovery supporting toxicity prediction. Here, we applied metabolomics profiling to cells treated with four well-selected SiO2 variants, differing in structure, size and surface charge. TiO2 NM-105 served as a benchmark. Responses were studied in vitro in rat lung epithelial cells (RLE-6TN) and alveolar macrophages (NR8383) and compared to in vivo responses in rat lung tissues obtained from in vivo instillation and short-term inhalation studies (STIS). Time- and concentration-dependent changes were observed in both in vitro models but with cell-type specific responses. Overall, the levels of lipids and biogenic amines (BAs) tended to increase in epithelial cells but decreased in macrophages. Many identified metabolites like Met-SO, hydroxy-Pro and spermidine were related to oxidative stress, indicating that oxidative stress contributes to the MoA for the selected NMs. Several biomarker candidates such as Asp, Asn, Ser, Pro, spermidine, putrescine and LysoPCaC16:1 were identified in vitro and verified in vivo. In this study, we successfully applied a metabolomics workflow for in vitro and in vivo samples, which proved to be well suited to identify potential biomarkers, to gain insights into NM structure–activity relationship and into the underlying MoA. [ABSTRACT FROM AUTHOR]

Regarding carcinogenicity testing, the long‐term rodent bioassay (RCB) has been the test required by most regulatory agencies across the world. Nonetheless, due to the lack of knowledge about its specificity, it has been argued that the RCB is unspecific or even invalid. Because of the substantial limitations of epidemiology to identify chemicals probably not carcinogenic to humans (PNCH), it has been very difficult to address the specificity of the RCB. Nevertheless, because mechanistic/pharmacological data are currently recognized as a valid stream of evidence for the identification of chemical hazards, the road is now open to gain insight into the specificity of the RCB. Based on sound mechanistic/pharmacological data that support the classification of chemicals as PNCH, 100 PNCH substances were gathered in this investigation. Contrary to what was previously forecast, in this study, the RCB exhibited a functional specificity that ranged from 83% to 91%, depending on the settings of the testing (2‐species vs. rats only, and the nominal maximum tolerated dose). Other contributions of this work were: (a) enabling the comparison, in terms of specificity, between the RCB and the alternative methods that could replace it (eg, Tg.AC mouse, rasH2 mouse); (b) disclosing what the specificity is for alternative methods that were developed using the RCB as the reference standard; and (c) expanding the previous narrow (only seven substances) set of chemicals identified as not likely to be carcinogenic to humans by hazard identification programs. Provided that mechanistic data are currently recognized as a valid stream of evidence, the road is open to achieve insight into the specificity of the long‐term rodent bioassay (RCB). Based on mechanistic or pharmacological data that supported the classification of chemicals as PNCH, 100 PNCH substances were gathered. Contrary to what was previously forecast, the RCB exhibited a functional specificity that ranged from 83% to 91%, depending on the settings of the testing (2‐species vs. rats, and the nominal maximum tolerated dose). [ABSTRACT FROM AUTHOR]

Engineered Nanomaterials (NMs), such as Superparamagnetic Iron Oxide Nanoparticles (SPIONs), offer significant benefits in a wide range of applications, including cancer diagnostic and therapeutic strategies. However, the use of NMs in biomedicine raises safety concerns due to lack of knowledge on possible biological interactions and effects. The initial basis for using SPIONs as biomedical MRI contrast enhancement agents was the idea that they are selectively taken up by macrophage cells, and not by the surrounding cancer cells. To investigate this claim, we analyzed the uptake of SPIONs into well-established cancer cell models and benchmarked this against a common macrophage cell model. In combination with fluorescent labeling of compartments and siRNA silencing of various proteins involved in common endocytic pathways, the mechanisms of internalization of SPIONs in these cell types has been ascertained utilizing reflectance confocal microscopy. Caveolar mediated endocytosis and macropinocytosis are both implicated in SPION uptake into cancer cells, whereas in macrophage cells, a clathrin-dependant route appears to predominate. Colocalization studies confirmed the eventual fate of SPIONs as accumulation in the degradative lysosomes. Dissolution of the SPIONs within the lysosomal environment has also been determined, allowing a fuller understanding of the cellular interactions, uptake, trafficking and effects of SPIONs within a variety of cancer cells and macrophages. Overall, the behavior of SPIONS in non-phagocytotic cell lines is broadly similar to that in the specialist macrophage cells, although some differences in the uptake patterns are apparent. [ABSTRACT FROM AUTHOR]

Adverse outcome pathways (AOPs) have been recently introduced as tools to map the mechanisms underlying toxic events relevant for chemical risk assessment. AOPs particularly depict the linkage between a molecular initiating event and an adverse outcome through a number of intermediate key events. An AOP has been previously introduced for cholestatic liver injury. The objective of this study was to test the robustness of this AOP for different types of cholestatic insult and the in vitro to in vivo extrapolation. For this purpose, in vitro samples from human hepatoma HepaRG cell cultures were exposed to cholestatic drugs (i.e. intrahepatic cholestasis), while in vivo samples were obtained from livers of cholestatic mice (i.e. extrahepatic cholestasis). The occurrence of cholestasis in vitro was confirmed through analysis of bile transporter functionality and bile acid analysis. Transcriptomic analysis revealed inflammation and oxidative stress as key events in both types of cholestatic liver injury. Major transcriptional differences between intrahepatic and extrahepatic cholestatic liver insults were observed at the level of cell death and metabolism. Novel key events identified by pathway analysis included endoplasmic reticulum stress in intrahepatic cholestasis, and autophagy and necroptosis in both intrahepatic as extrahepatic cholestasis. This study demonstrates that AOPs constitute dynamic tools that should be frequently updated with new input information. [ABSTRACT FROM AUTHOR]

Toxicology and related concepts are being taught within chemistry education programs to enable students to (1) develop an awareness of toxicological principles and concepts that are otherwise absent from the chemistry curriculum for undergraduates and (2) design chemical products and processes that have reduced health and environmental hazards. This manuscript presents three model courses from different higher education institutions, presenting different approaches for introducing toxicology concepts to students. The models are presented not as prescriptive approaches, but as inspirational models for others to use to generate their own unique approaches to integrating and teaching toxicology concepts.

Drug-induced liver injury (DILI) is a major problem for pharmaceutical industry and drug development. Mechanisms of DILI are many and varied. Elucidating the mechanisms of DILI will allow clinicians to prevent liver failure, need for liver transplantation, and death induced by drugs. Methimazole and propylthiouracil (PTU) are two convenient antithyroid agents which their administration is accompanied by hepatotoxicity as a deleterious side effect. Although several cases of antithyroid drugs-induced liver injury are reported, there is no clear idea about the mechanism(s) of hepatotoxicity induced by these medications. Different mechanisms such as reactive metabolites formation, oxidative stress induction, intracellular targets dysfunction, and immune-mediated toxicity are postulated to be involved in antithyroid agents-induced hepatic damage. Due to the idiosyncratic nature of antithyroid drugs-induced hepatotoxicity, it is impossible to draw a specific conclusion about the mechanisms of liver injury. However, it seems that reactive metabolite formation and immune-mediated toxicity have a great role in antithyroids liver toxicity, especially those caused by methimazole. This review attempted to discuss different mechanisms proposed to be involved in the hepatic injury induced by antithyroid drugs.

Experimental modeling to identify specific inhalation hazards for nanomaterials has in the main focused on in vivo approaches. However, these models suffer from uncertainties surrounding species-specific differences and cellular targets for biologic response. In terms of pulmonary exposure, approaches which combine ‘inhalation-like’ nanoparticulate aerosol deposition with relevant human cell and tissue air-liquid interface cultures are considered an important complement to in vivo work. In this study, we utilized such a model system to build on previous results from in vivo exposures, which highlighted the small airway epithelium as a target for silver nanoparticle (AgNP) deposition. RNA-SEQ was used to characterize alterations in mRNA and miRNA within the lung. Organotypic-reconstituted 3D human primary small airway epithelial cell cultures (SmallAir) were exposed to the same spark-generated AgNP and at the same dose used in vivo, in an aerosol-exposure air-liquid interface (AE-ALI) system. Adverse effects were characterized using lactate, LDH release and alterations in mRNA and miRNA. Modest toxicological effects were paralleled by significant regulation in gene expression, reflective mainly of specific inflammatory events. Importantly, there was a level of concordance between gene expression changes observed in vitro and in vivo. We also observed a significant correlation between AgNP and mass equivalent silver ion (Ag+) induced transcriptional changes in SmallAir cultures. In addition to key mechanistic information relevant for our understanding of the potential health risks associated with AgNP inhalation exposure, this work further highlights the small airway epithelium as an important target for adverse effects. [ABSTRACT FROM AUTHOR]

A variety of experimental and epidemiological studies lend support to the Developmental Origin of Health and Disease (DOHaD) concept. Yet, the actual mechanisms accounting for mid- and long-term effects of early-life exposures remain unclear. Epigenetic alterations such as changes in DNA methylation, histone modifications and the expression of certain RNAs have been suggested as possible mediators of long-term health effects of environmental stressors. This report captures discussions and conclusions debated during the last Prenatal Programming and Toxicity meeting held in Japan. Its first aim is to propose a number of criteria that are critical to support the primary contribution of epigenetics in DOHaD and intergenerational transmission of environmental stressors effects. The main criteria are the full characterization of the stressors, the actual window of exposure, the target tissue and function, the specificity of the epigenetic changes and the biological plausibility of the linkage between those changes and health outcomes. The second aim is to discuss long-term effects of a number of stressors such as smoking, air pollution and endocrine disruptors in order to identify the arguments supporting the involvement of an epigenetic mechanism. Based on the developed criteria, missing evidence and suggestions for future research will be identified. The third aim is to critically analyze the evidence supporting the involvement of epigenetic mechanisms in intergenerational and transgenerational effects of environmental exposure and to particularly discuss the role of placenta and sperm. While the article is not a systematic review and is not meant to be exhaustive, it critically assesses the contribution of epigenetics in the long-term effects of environmental exposures as well as provides insight for future research. [ABSTRACT FROM AUTHOR]

Marine mammals, due to their long life span, key position in the food web, and large lipid deposits, often face significant health risks from accumulating contaminants. This systematic review examines published literature on pollutant-induced adverse health effects in the International Union for Conservation of Nature (IUCN) red-listed marine mammal species. Thereby, identifying gaps in literature across different extinction risk categories, spatial distribution and climatic zones of studied habitats, commonly used methodologies, researched pollutants, and mechanisms from cellular to population levels. Our findings reveal a lower availability of exposure-effect data for higher extinction risk species (critically endangered 16%, endangered 15%, vulnerable 66%), highlighting the need for more research. For many threatened species in the Southern Hemisphere pollutant-effect relationships are not established. Non-destructively sampled tissues, like blood or skin, are commonly measured for exposure assessment. The most studied pollutants are POPs (31%), metals (30%), and pesticides (17%). Research on mixture toxicity is scarce while pollution-effect studies primarily focus on molecular and cellular levels. Bridging the gap between molecular data and higher-level effects is crucial, with computational approaches offering a high potential through in vitro to in vivo extrapolation using (toxico-)kinetic modelling. This could aid in population-level risk assessment for threatened marine mammals. [Display omitted] • The higher the extinction risk, the fewer exposure-effect data are available. • Lack of studies in the Southern Hemisphere shows a spatial bias in the literature. • Commonly studied pollutants are persistent organic pollutants, metals, pesticides. • Pollution-effect studies focus on molecular and cellular levels. • In silico and in vitro approaches aid in assessing in vivo effects. [ABSTRACT FROM AUTHOR]

[Display omitted] Adverse outcome pathway (AOP) networks are versatile tools in toxicology and risk assessment that capture and visualize mechanisms driving toxicity originating from various data sources. They share a common structure consisting of a set of molecular initiating events and key events, connected by key event relationships, leading to the actual adverse outcome. AOP networks are to be considered living documents that should be frequently updated by feeding in new data. Such iterative optimization exercises are typically done manually, which not only is a time-consuming effort, but also bears the risk of overlooking critical data. The present study introduces a novel approach for AOP network optimization of a previously published AOP network on chemical-induced cholestasis using artificial intelligence to facilitate automated data collection followed by subsequent quantitative confidence assessment of molecular initiating events, key events, and key event relationships. Artificial intelligence-assisted data collection was performed by means of the free web platform Sysrev. Confidence levels of the tailored Bradford-Hill criteria were quantified for the purpose of weight-of-evidence assessment of the optimized AOP network. Scores were calculated for biological plausibility, empirical evidence, and essentiality, and were integrated into a total key event relationship confidence value. The optimized AOP network was visualized using Cytoscape with the node size representing the incidence of the key event and the edge size indicating the total confidence in the key event relationship. This resulted in the identification of 38 and 135 unique key events and key event relationships, respectively. Transporter changes was the key event with the highest incidence, and formed the most confident key event relationship with the adverse outcome, cholestasis. Other important key events present in the AOP network include: nuclear receptor changes, intracellular bile acid accumulation, bile acid synthesis changes, oxidative stress, inflammation and apoptosis. This process led to the creation of an extensively informative AOP network focused on chemical-induced cholestasis. This optimized AOP network may serve as a mechanistic compass for the development of a battery of in vitro assays to reliably predict chemical-induced cholestatic injury. [ABSTRACT FROM AUTHOR]

Toxicology and related concepts are being taught within chemistry education programs to enable students to (1) develop an awareness of toxicological principles and concepts that are otherwise absent from the chemistry curriculum for undergraduates and (2) design chemical products and processes that have reduced health and environmental hazards. This manuscript presents three model courses from different higher education institutions, presenting different approaches for introducing toxicology concepts to students. The models are presented not as prescriptive approaches, but as inspirational models for others to use to generate their own unique approaches to integrating and teaching toxicology concepts. [ABSTRACT FROM PUBLISHER]

This study presents a mechanistic model for identifying oxide nanoparticles that induce a high level of neutrophils in the bronchoalveolar lavage fluid, an important marker for lung inflammogenicity. The model is based on 4 nanoparticles' physicochemical properties, ie, the reactivity, surface charge, wettability, and dissolution. First, I calculate these properties and show that theoretical values reproduce acceptably the experimental measurements. Then, I combine these properties with mechanistic knowledge to build a classification model for the prediction of acute in vivo lung inflammogenicity, measured as the total number of polymorphonuclear neutrophils. The model uses reactivity and dissolution properties of nanoparticles as toxicological initiating events, whereas surface charge and wettability are characteristics involved in the interactions between the nanoparticles and the lung surfactant, eventually leading to increased cellular uptake and bioaccumulation. The model is validated on a set of 43 oxide nanoparticles tested in vivo to confirm that acute lung inflammation can be described using this mechanistic framework. In addition, I also develop a linear regression model for insoluble nanoparticles to quantitatively predict the polymorphonuclear neutrophil count as a function of reactivity and surface charge. The proposed models are based on mechanistic knowledge and can support the development of adverse outcome pathways, risk assessment frameworks and safe design strategies at early stages of material's R&D. [ABSTRACT FROM AUTHOR]

Risk assessors use liver endpoints in rodent toxicology studies to assess the safety of chemical exposures. Yet, rodent endpoints may not accurately reflect human responses. For this reason and others, human-based in vitro models are being developed and anchored to adverse outcome pathways to better predict adverse human health outcomes. Here, a networked adverse outcome pathway-guided selection of biology-based assays for lipid uptake, lipid efflux, fatty acid oxidation, and lipid accumulation were developed. These assays were evaluated in a metabolically competent human hepatocyte cell model (HepaRG) exposed to compounds known to cause steatosis (amiodarone, cyclosporine A, and T0901317) or activate lipid metabolism pathways (troglitazone, Wyeth-14,643, and 22(R)-hydroxycholesterol). All of the chemicals activated at least one assay, however, only T0901317 and cyclosporin A dose-dependently increased lipid accumulation. T0901317 and cyclosporin A increased fatty acid uptake, decreased lipid efflux (inferred from apolipoprotein B100 levels), and increased fatty acid synthase protein levels. Using this biologically-based evaluation of key events regulating hepatic lipid levels, we demonstrated dysregulation of compensatory pathways that normally balance hepatic lipid levels. This approach may provide biological plausibility and data needed to increase confidence in linking in vitrobased measurements to chemical effects on adverse human health outcomes. [ABSTRACT FROM AUTHOR]

This study investigated the role of nanoparticle size and surface chemistry on biocorona composition and its effect on uptake, toxicity and cellular responses in human umbilical vein endothelial cells (HUVEC), employing 40 and 80 nm gold nanoparticles (AuNP) with branched polyethyleneimine (BPEI), lipoic acid (LA) and polyethylene glycol (PEG) coatings. Proteomic analysis identified 59 hard corona proteins among the various AuNP, revealing largely surface chemistry-dependent signature adsorbomes exhibiting human serum albumin (HSA) abundance. Size distribution analysis revealed the relative instability and aggregation inducing potential of bare and corona-bound BPEI-AuNP, over LA- and PEG-AuNP. Circular dichroism analysis showed surface chemistry-dependent conformational changes of proteins binding to AuNP. Time-dependent uptake of bare, plasma corona (PC) and HSA corona-bound AuNP (HSA-AuNP) showed significant reduction in uptake with PC formation. Cell viability studies demonstrated dose-dependent toxicity of BPEI-AuNP. Transcriptional profiling studies revealed 126 genes, from 13 biological pathways, to be differentially regulated by 40 nm bare and PC-bound BPEI-AuNP (PC-BPEI-AuNP). Furthermore, PC formation relieved the toxicity of cationic BPEI-AuNP by modulating expression of genes involved in DNA damage and repair, heat shock response, mitochondrial energy metabolism, oxidative stress and antioxidant response, and ER stress and unfolded protein response cascades, which were aberrantly expressed in bare BPEI-AuNP-treated cells. NP surface chemistry is shown to play the dominant role over size in determining the biocorona composition, which in turn modulates cell uptake, and biological responses, consequently defining the potential safety and efficacy of nanoformulations. [ABSTRACT FROM AUTHOR]

Pharmaceuticals have been classified as emerging contaminants in the aquatic ecosystem, mainly due to their increased use and improper disposal. A significant range of pharmaceutical compounds and their metabolites have been globally detected in surface waters and pose detrimental effects to non-target organisms. Monitoring pharmaceutical water pollution relies on the analytical approaches for their detection, however, such approaches are limited by their sensitivity limit and coverage of the wide range pharmaceutical compounds. This lack of realism in risk assessment is bypassed with effect-based methods, which are complemented by chemical screening and impact modelling, and are able to provide mechanistic insight for pollution. Focusing on the freshwater ecosystem, in this study we evaluated the acute effects on daphnids for three distinct groups of pharmaceuticals; antibiotics, estrogens, and a range of commonly encountered environmentally relevant pharmaceutical pollutants. Combining several endpoints such as mortality, biochemical (enzyme activities) and holistic (metabolomics) we discovered distinct patterns in biological responses. In this study, changes in enzymes of metabolism e.g. phosphatases and lipase, as well as the detoxification enzyme, glutathione-S-transferase, were recorded following acute exposure to the selected pharmaceuticals. A targeted analysis of the hydrophilic profile of daphnids revealed mainly the up-regulation of metabolites following metformin, gabapentin, amoxicillin, trimethoprim and β-estradiol. Whereas gemfibrozil, sulfamethoxazole and oestrone exposure resulted in the down-regulation of majority of metabolites. • Monitoring pharmaceutical pollution requires sensitive novel methods and metrics. • Daphnids act as a "canary in the coal mine" for water pollution. • Molecular endpoints and sensitive metabolomics provide mechanistic insight for the impact of pharmaceutical pollutants. [ABSTRACT FROM AUTHOR]

Titanium dioxide nanoparticles (TiO2-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1–50 μg/mL TiO2-NPs, 86% anatase/14% rutile, 24 ± 6nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO2-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO2-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO2-NPs. [ABSTRACT FROM AUTHOR]

Hepatic steatosis is a condition were fat accumulates in the liver and it is associated with extra-hepatic diseases related to metabolic syndrome and systemic energy metabolism. If not reversed, steatosis can progress to steatohepatitis and irreversible stages of liver disease including fibrosis, cirrhosis, hepatocellular carcinoma, and death. From a public health standpoint, identifying chemical exposures that may be factors in steatosis etiology are important for preventing hepatotoxicity and liver disease progression. It is therefore important to identify the biological events that are key for steatosis pathology mediated by chemical exposure. In this review, we give a current overview of the complex biological cascades that can disrupt lipid homeostasis in hepatocytes in the context of 4 apical key events central to hepatic lipid retention: hepatic fatty acid (FA) uptake, de novo FA and lipid synthesis, FA oxidation, and lipid efflux. Our goal is to review these key cellular events and visually summarize them using a network for application in pathway-based toxicity testing. This effort provides a foundation to improve next-generation chemical screening efforts that may be used to prevent and ultimately reverse the growing incidence of fatty liver disease in our population. [ABSTRACT FROM AUTHOR]

With rapid development of nanotechnology, quantum dots (QDs) as advanced nanotechnology products have been widely used in biological and biomedical studies, including neuroscience, due to their superior optical properties. In recent years, there has been intense concern regarding the toxicity of QDs with a growing number of studies. However, the knowledge of neurotoxic consequences of QDs applied in living organisms is lagging behind their development, while a potential risk of neurotoxicity arises if mass production of QDs leads to increased exposure and distribution in the nervous system. Owing to the quantum size effect of QDs, they are capable of crossing the blood-brain barrier or moving along neural pathways and entering the brain. Nevertheless, the interactions of QDs with cells and tissues in the central nervous system are not well understood. This review highlighted research advances on the neurotoxicity of QDs in the central nervous system, including oxidative stress injury, elevated cytoplasmic Ca2+ levels and autophagy to damage in vitro neural cells, and impairments of synaptic transmission and plasticity as well as brain functions in tested animals, with the hope of throwing light on future research directions of QD neurotoxicity, which is a demanding topic that requires further exploration. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Pesticides are toxic and biological substances used for mitigating harmful pests. Their application in agricultural fields and homes increased environmental pollution. Studies showed their harmful effects on human health, specifically children, who are more vulnerable than adults. The International Agency for Research on Cancer (IARC) has introduced several pesticides as carcinogens. This study aims to systematically summarize and review all studies related to pesticides and cancer. This systematic review is based on PRISMA rules. Three central databases were employed to find studies on pesticide exposure and cancer correlation published from 2017 to September 2022. After reviewing several studies, we found that most studies revealed a significant relationship between pesticide exposure and an increased cancer incidence rate. Among the most studied group of pesticides is organochlorine (OC) pesticides. OC pesticides and their residues could significantly increase cancer in children and adults. Mechanistic studies revealed that pesticides could increase the risk of different cancers by genetics, like an increased expression of some genes like p21, p53 or epigenetic impairments. Cell cycle impairments like expanding the G1 to S phase transition are another mechanism of causing cancer. DNA methylation and histone modifications increase the risk of numerous cancers. Based on epidemiological studies, pesticides are a significant concern to human health, specifically cancer development, and should be more restrained. Their most reported mechanism of action were genetic and epigenetic impairments which cause cancers. • This study exhibited a significant relationship between pesticides exposure and an increased cancer incidence rate. • Organochlorine pesticides and their residues increased many types of cancer incidence significantly. However, Organophosphorus results were controversial. • Different types of cancers increased in children whose parents were exposed to pesticides during pregnancy. • Mechanistic studies revealed that pesticides could increase the risk of different types of cancers by genetic and epigenetic impairments. However, these components induce apoptosis and autophagy, cancer-protective mechanisms. [ABSTRACT FROM AUTHOR]

Skin sensitization has been described as an adverse outcome pathway (AOP), comprising a number of molecular events leading to the final adverse effect. In a new paradigm of toxicology, attempts are made to collect information using single mechanistic tests addressing different targets along such an AOP and to then integrate this information to arrive at a final toxicological prediction. This proposal is strongly influenced by the availability of methods for high-throughput screening of cellular events. Reporter cell lines are a particularly useful tool in such screening paradigms, as they can deliver highly reproducible and easily measureable results, and they can be designed to quantify induction or suppression at the transcription level of very specific molecular targets within cells. The first cell-based assay for skin sensitization, which has recently received ECVAM and OECD endorsement, is the reporter cell assay KeratinoSens™, reflecting activation of the Nrf2 pathway, and other assays measuring the Nrf2 pathway are under development or validation. An alternative approach (THP-G8) was recently developed based on activation of the Interleukin-8 gene. Here, we review these assays, their role in the AOP, their mechanistic interrelationships, their use for hazard and risk assessment, and their application in integrated testing strategies. At the same time, this study reviews (1) other cellular markers for sensitizers, and the potential to develop new reporter gene assays providing additional, non-redundant information, and (2) it presents approaches and new experimental data on attempts to further improve the predictivity of the existing assay. [ABSTRACT FROM AUTHOR]

The inhalation of zinc oxide engineered nanomaterials (ENMs) has been linked to cardiorespiratory dysfunction in mammalian models but the effects of aquatic ENM exposure on fish have not been fully investigated. Nano-zinc oxide (nZnO) is widely used in consumer products such as sunscreens and can make its way into aquatic ecosystems from domestic and commercial wastewater. This study examined the impact of an environmentally relevant nZnO formulation on cardiorespiratory function and energy metabolism in the white sucker (Catostomus commersonii), a freshwater teleost fish. Evidence of oxidative and cellular stress was present in gill tissue, including increases in malondialdehyde levels, heat shock protein (HSP) expression, and caspase 3/7 activity. Gill Na+/K+-ATPase activity was also higher by approximately three-fold in nZnO-treated fish, likely in response to increased epithelial permeability or structural remodeling. Despite evidence of toxicity in gill, plasma cortisol and lactate levels did not change in animals exposed to 1.0 mg L−1 nZnO. White suckers also exhibited a 35% decrease in heart rate during nZnO exposure, with no significant changes in resting oxygen consumption or tissue energy stores. Our results suggest that tissue damage or cellular stress resulting from nZnO exposure activates gill neuroepithelial cells, triggering a whole-animal hypoxic response. An increase in parasympathetic nervous signaling will decrease heart rate and may reduce energy demand, even in the face of an environmental toxicant. We have shown that acute exposure to nZnO is toxic to white suckers and that ENMs have the potential to negatively impact cardiorespiratory function in adult fish. [ABSTRACT FROM AUTHOR]

CuO NPs have previously been reported as toxic to a range of cell cultures including kidney epithelial cells from the frog, Xenopus laevis (A6). Here we examine the molecular mechanisms affecting toxicity of Cu in different forms and particle sizes. A6 cells were exposed to ionic Cu (Cu 2+ ) or CuO particles of three different sizes: CuO NPs of 6 nm (NP6), larger Poly-dispersed CuO NPs of <100 nm (Poly) and CuO Micro particles of <5 μm (Micro), at 200 μM, equal to 12.7 mg Cu/L. Poly was significantly more toxic than NP6, Micro and Cu 2+ to A6 cells, causing DNA damage, decreased cell viability and levels of reduced glutathione (GSH) and eventually cell death. We show that ROS (Reactive Oxygen Species) generation plays a key role and occurs early in Poly toxicity as Poly-induced DNA damage and cell death could be mitigated by the antioxidant NAC (N-acetyl-cysteine). Here we propose a model of the sequence of events explaining Poly toxicity. Briefly, the events include: cellular uptake, most likely via endocytosis, production of ROS, which cause DNA damage that activates a signaling pathway which eventually leads to cell death, mainly via apoptosis. [ABSTRACT FROM AUTHOR]

A weak aspect of the in vitro studies devoted to get information on the toxic, genotoxic and carcinogenic properties of nanomaterials is that they are usually conducted under acute-exposure and high-dose conditions. This makes difficult to extrapolate the results to human beings. To overcome this point, we have evaluated the cell transforming ability of cobalt nanoparticles (CoNPs) after long-term exposures (12 weeks) to sub-toxic doses (0.05 and 0.1 µg/mL). To get further information on whether CoNPs-induced oxidative DNA damage is relevant for CoNPs carcinogenesis, the cell lines selected for the study were the wild-type mouse embryonic fibroblast (MEF Ogg1+/+) and its isogenic Ogg1 knockout partner (MEF Ogg1−/−), unable to properly eliminate the 8-OH-dG lesions from DNA. Our initial short-term exposure experiments demonstrate that low doses of CoNPs are able to induce reactive oxygen species (ROS) and that MEF Ogg1−/− cells are more sensitive to CoNPs-induced acute toxicity and oxidative DNA damage. On the other hand, long-term exposures of MEF cells to sub-toxic doses of CoNPs were able to induce cell transformation, as indicated by the observed morphological cell changes, significant increases in the secretion of metalloproteinases (MMPs) and anchorage-independent cell growth ability, all cancer-like phenotypic hallmarks. Interestingly, such changes were significantly dependent on the cell line used, the Ogg1−/− cells being particularly sensitive. Altogether, the data presented here confirms the potential carcinogenic risk of CoNPs and points out the relevance of ROS and Ogg1 genetic background on CoNPs-associated effects. [ABSTRACT FROM AUTHOR]

Drug-induced liver injury (DILI) is a major problem for pharmaceutical industry and drug development. Mechanisms of DILI are many and varied. Elucidating the mechanisms of DILI will allow clinicians to prevent liver failure, need for liver transplantation, and death induced by drugs. Methimazole and propylthiouracil (PTU) are two convenient antithyroid agents which their administration is accompanied by hepatotoxicity as a deleterious side effect. Although several cases of antithyroid drugs-induced liver injury are reported, there is no clear idea about the mechanism(s) of hepatotoxicity induced by these medications. Different mechanisms such as reactive metabolites formation, oxidative stress induction, intracellular targets dysfunction, and immune-mediated toxicity are postulated to be involved in antithyroid agents-induced hepatic damage. Due to the idiosyncratic nature of antithyroid drugs-induced hepatotoxicity, it is impossible to draw a specific conclusion about the mechanisms of liver injury. However, it seems that reactive metabolite formation and immune-mediated toxicity have a great role in antithyroids liver toxicity, especially those caused by methimazole. This review attempted to discuss different mechanisms proposed to be involved in the hepatic injury induced by antithyroid drugs. [ABSTRACT FROM AUTHOR]

Although toxic effects of zinc oxide nanoparticles (ZnO NPs) have been previously studied, there are still controversies in terms of dose, size, shape, and affecting cells. By such a perspective, in this study, small size of ZnO NPs with a diameter of 10 nm at low concentrations was studied for any effect on the viability and function of isolated rat pancreatic islets. Islets of Langerhans were isolated and assessed for viability, functionality (insulin secretion), cytosolic reactive oxygen species (ROS), and apoptosis by flow cytometry. The LC of ZnO NPs was found at 1,400 ng/mL at the first phase of the study. A meaningful increase in viability of islets and insulin secretion in basal and even stimulated concentrations of glucose was found by ZnO NPs (70 ng/mL) with p < 0.001 and p < 0.05, respectively. Likewise, ZnO NPs in 70 ng/mL concentration decreased cytosolic ROS generation ( p < 0.05). In the meantime, the percentage of early stage of apoptotic cells dropped down to 17 % (from 29 % of control). These results for the first time confirm that ZnO NPs are not only safe when used at dose of 70 ng/mL but also improve viability and function of pancreatic islets and meanwhile reduce oxidative stress and prevent cells from entering the apoptotic phase. [ABSTRACT FROM AUTHOR]

Abstract: The selection of drug candidates early in development has become increasingly important to minimize the use of animals and to avoid costly failures of drugs later in development. In vitro systems to predict and assess organ toxicity have so far been of limited value due to difficulties in demonstrating in vivo-relevant toxicity at a cell culture level. To overcome the limitations of single-cell type monolayer cultures and short-lived primary cell preparations, researchers have created novel 3-dimensional culture systems which appear to more closely resemble in vivo biology. These could become a key for the pharmaceutical industry in the evaluation of drug candidates. However, the value and acceptance of those new models in standard drug safety applications have yet to be demonstrated. This review aims to provide an overview of the different approaches undertaken in the field of pre-clinical safety assessment, organ toxicity, in particular, with an emphasis on examples and technical challenges. [Copyright &y& Elsevier]

Muscle dysfunction in acute organophosphorus (OP) poisoning is a cause of death in human. The present study was conducted to identify the mechanism of action of OP in terms of muscle mitochondrial dysfunction. Electromyography (EMG) was conducted on rats exposed to the acute oral dose of malathion (400 mg/kg) that could inhibit acetylcholinesterase activity up to 70%. The function of mitochondrial respiratory chain and the rate of production of reactive oxygen species (ROS) from intact mitochondria were measured. The bioenergetic pathways were studied by measurement of adenosine triphosphate (ATP), lactate, and glycogen. To identify mitochondrial-dependent apoptotic pathways, the messenger RNA (mRNA) expression of bax and bcl-2, protein expression of caspase-9, mitochondrial cytochrome c release, and DNA damage were measured. The EMG confirmed muscle weakness. The reduction in activity of mitochondrial complexes and muscular glycogen with an elevation of lactate was in association with impairment of cellular respiration. The reduction in mitochondrial proapoptotic stimuli is indicative of autophagic process inducing cytoprotective effects in the early stage of stress. Downregulation of apoptotic signaling may be due to reduction in ATP and ROS, and genotoxic potential of malathion. The maintenance of mitochondrial integrity by means of artificial electron donors and increasing exogenous ATP might prevent toxicity of OPs. [ABSTRACT FROM PUBLISHER]

Highlights: [•] The mechanisms of mercury toxicity towards skeletal muscles is poorly understood. [•] Glycogen phosphorylase (GP) plays a key role in skeletal muscle functions. [•] Mercury irreversibly inhibits the activity of GP in muscle cells and tissues. [•] Inhibition is due to covalent reaction of mercury with cysteine residues of GP. [•] Inhibition of GP by mercury may contribute to toxicity of mercury in muscles. [Copyright &y& Elsevier]

Abstract: Adverse outcome pathways (AOPs) are novel tools in toxicology and human risk assessment with broad potential. AOPs are designed to provide a clear-cut mechanistic representation of critical toxicological effects that span over different layers of biological organization. AOPs share a common structure consisting of a molecular initiating event, a series of intermediate steps and key events, and an adverse outcome. Development of AOPs ideally complies with OECD guidelines. This also holds true for AOP evaluation, which includes consideration of the Bradford Hill criteria for weight-of-evidence assessment and meeting a set of key questions defined by the OECD. Elaborate AOP frameworks have yet been proposed for chemical-induced skin sensitization, cholestasis, liver fibrosis and liver steatosis. These newly postulated AOPs can serve a number of ubiquitous purposes, including the establishment of (quantitative) structure–activity relationships, the development of novel in vitro toxicity screening tests and the elaboration of prioritization strategies. [Copyright &y& Elsevier]

Background: Carbon nanotubes (CNT) are fibre-like nanomaterials whose structural similarity to asbestos has raised concerns that they may also pose a mesothelioma hazard. The objective of this study was to examine the inflammatory potential of three CNT samples of differing length on the lungs and pleural cavity following introduction into the airspaces of mice. ResultsAspiration of the two short/tangled and one long CNT sample into the lungs of mice resulted in a length-dependent inflammatory response at 1 week, i.e., only the long CNT sample caused acute neutrophilic inflammation in bronchoalveolar lavage at 1 week and progressive thickening of the alveolar septa. The authors also report length-dependent inflammatory responses in the pleural lavage after exposure only to the long CNT. The inflammatory response in the pleural cavity to long fibres and the appearance of lesions along the chest wall and diaphragm was not present at 1 week and only evident by 6 weeks post-exposure. Conclusion: Length-dependent pathogenicity is a feature of asbestos and the results presented in this study demonstrate similar length-dependent pathogenicity of CNT in the lungs and pleural space following airspace deposition. The data support the contention that long CNT reach the pleura from the airspaces, and that they are retained at the parietal pleura and cause inflammation and lesion development. The parietal pleura is the site of origin of mesothelioma and inflammation is considered to be a process involved in asbestos carcinogenesis and so the data support the contention that CNT may pose an asbestos-like mesothelioma hazard. [ABSTRACT FROM AUTHOR]

Allergic asthma is a chronic inflammatory disorder of the airway associated with bronchial obstruction, airway hyper-reactivity (AHR), and mucus production. The epithelium may direct and propagate asthmatic-like responses. Central to this theory is the observation that viruses, air pollution, and allergens promote epithelial damage and trigger the generation of IL-25, IL-33, and TSLP via innate pathways such as TLRs and purinergic receptors. Similarly, engineered nanomaterials promote a Th2-associated pathophysiology. In this study, we tested the hypothesis that instillation of multi-walled carbon nanotubes (MWCNT) impair pulmonary function in C57Bl/6 mice due to the development of IL-33-dependent Th2-associated inflammation. MWCNT exposure resulted in elevated levels of IL-33 in the lavage fluid (likely originating from airway epithelial cells), enhanced AHR, eosinophil recruitment, and production of Th2-associated cytokines and chemokines. Moreover, these events were dependent on IL-13 signaling and the IL-33/ST2 axis, but independent of T and B cells. Finally, MWCNT exposure resulted in the recruitment of innate lymphoid cells. Collectively, our data suggest that MWCNT induce epithelial damage that results in release of IL-33, which in turn promotes innate lymphoid cell recruitment and the development of IL-13-dependent inflammatory response. [ABSTRACT FROM AUTHOR]

The use of nanoparticles in consumer products increases their prevalence in the environment and the potential risk to human health. Although recent studies have shown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a more detailed view of the underlying mechanisms of this response needs to be established. Here the effects of nano-TiO2 on the DNA damage response and DNA replication dynamics were investigated in human dermal fibroblasts. Specifically, the relationship between nano-TiO2 and the DNA damage response pathways regulated by ATM/Chk2 and ATR/Chk1 were examined. The results show increased phosphorylation of H2AX, ATM, and Chk2 after exposure. In addition, nano-TiO2 inhibited the overall rate of DNA synthesis and frequency of replicon initiation events in DNA combed fibers. Taken together, these results demonstrate that exposure to nano-TiO2 activates the ATM/Chk2 DNA damage response pathway. [ABSTRACT FROM AUTHOR]

Surfactant protein D (SP-D) is primarily expressed in the lungs and modulates pro- and anti-inflammatory processes to toxic challenge, maintaining lung homeostasis. We investigated the interaction between NPs and SP-D and subsequent uptake by cells involved in lung immunity. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) measured NP aggregation, particle size and charge in native human SP-D (NhSP-D) and recombinant fragment SP-D (rfhSP-D). SP-D aggregated NPs, especially following the addition of calcium. Immunohistochemical analysis of A549 epithelial cells investigated the co-localization of NPs and rfhSP-D. rfhSP-D enhanced the co-localisation of NPs to epithelial A549 cells in vitro. NP uptake by alveolar macrophages (AMs) and lung dendritic cells (LDCs) from C57BL/6 and SP-D knock-out mice were compared. AMs and LDCs showed decreased uptake of NPs in SP-D deficient mice compared to wild-type mice. These data confirmed an interaction between SP-D and NPs, and subsequent enhanced NP uptake. [ABSTRACT FROM AUTHOR]

Abstract: Organophosphorous (OPs) pesticides are the most widely used pesticides in the agriculture and home. However, many acute or chronic poisoning reports about OPs have been published in the recent years. Mitochondria as a site of cellular oxygen consumption and energy production can be a target for OPs poisoning as a non-cholinergic mechanism of toxicity of OPs. In the present review, we have reviewed and criticized all the evidences about the mitochondrial dysfunctions as a mechanism of toxicity of OPs. For this purpose, all biochemical, molecular, and morphological data were retrieved from various studies. Some toxicities of OPs are arisen from dysfunction of mitochondrial oxidative phosphorylation through alteration of complexes I, II, III, IV and V activities and disruption of mitochondrial membrane. Reductions of adenosine triphosphate (ATP) synthesis or induction of its hydrolysis can impair the cellular energy. The OPs disrupt cellular and mitochondrial antioxidant defense, reactive oxygen species generation, and calcium uptake and promote oxidative and genotoxic damage triggering cell death via cytochrome C released from mitochondria and consequent activation of caspases. The mitochondrial dysfunction induced by OPs can be restored by use of antioxidants such as vitamin E and C, alpha-tocopherol, electron donors, and through increasing the cytosolic ATP level. However, to elucidate many aspect of mitochondrial toxicity of Ops, further studies should be performed. [Copyright &y& Elsevier]

The need for a more mechanistic understanding of the ways in which chemicals modulate biological pathways is urgent if we are to identify and better assess safety issues relating to a wide range of substances developed by the pharmaceutical, chemical, agri-bio, and cosmetic industries. Omics technologies provide a valuable opportunity to refine existing methods and provide information for so-called integrated testing strategies via the creation of signatures of toxicity. By mapping these signatures to underlying pathways of toxicity, some of which have been identified by toxicologists over the last few decades, and bringing them together with pathway information determined from biochemistry and molecular biology, a "systems toxicology" approach will enable virtual experiments to be conducted that can improve the prediction of hazard and the assessment of compound toxicity. [ABSTRACT FROM AUTHOR]

Ingested, inhaled or injected particles come into contact with biological fluids containing polymers, such as the protein fibrinogen. We studied interactions between well-characterized submicron particles or nanoparticles (NPs) and human fibrinogen. In vitro aggregation and zeta potential measurements of different sized and functionalized polystyrene, carbon black and silica NPs suspended in fibrinogen solutions were made. Particle size, surface charge and aggregation behaviour significantly changed in the presence of fibrinogen. Polymer (protein) bridging and bridge flocculation was observed. We concluded: (1) NP aggregation rate in a fibrinogen solution depended on particle surface type; (2) amine-functionalized particles aggregated more slowly in fibrinogen; and (3) particle morphology strongly influenced biologically available surface for protein attachment, but this did not correlate well with particle surface area for complex particles (calculated or measured). Interaction of particles and NPs with pro-coagulant polymers may therefore dictate the NP surface dose presentation to cells/organs and subsequent cellular effects, in and ex vivo. [ABSTRACT FROM AUTHOR]

We review the mechanisms and pathways whereby nanoparticles might cause genotoxicity. Primary and secondary mechanisms are discussed in relation to the general particle toxicology paradigm. We also discuss how we might improve genotoxicity assays for nanoparticles. In this context we describe the role of the dispersion and the protein corona, the most relevant metric, choice of controls and new endpoints for genotoxicity along with the need for a structure activity model of NP genotoxicity. [ABSTRACT FROM AUTHOR]

Abstract: A common animal model of chemical hepatocarcinogenesis was used to examine the utility of transcriptomic and proteomic data to identify early biomarkers related to chemically induced carcinogenesis. N-nitrosomorpholine, a frequently used genotoxic model carcinogen, was applied via drinking water at 120 mg/L to male Wistar rats for 7 weeks followed by an exposure-free period of 43 weeks. Seven specimens of each treatment group (untreated control and 120 mg/L N-nitrosomorpholine in drinking water) were sacrificed at nine time points during and after N-nitrosomorpholine treatment. Individual samples from the liver were prepared for histological and toxicogenomic analyses. For histological detection of preneoplastic and neoplastic tissue areas, sections were stained using antibodies against the placental form of glutathione-S-transferase (GST-P). Gene and protein expression profiles of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat gene chips and two-dimensional gel electrophoresis-based proteomics, respectively. In order to compare results obtained by histopathology, transcriptomics and proteomics, GST-P-stained liver sections were evaluated morphometrically, which revealed a parallel time course of the area fraction of preneoplastic lesions and gene plus protein expression patterns. On the transcriptional level, an increase of hepatic GST-P expression was detectable as early as 3 weeks after study onset. Comparing deregulated genes and proteins, eight species were identified which showed a corresponding expression profile on both expression levels. Functional analysis suggests that these genes and corresponding proteins may be useful as biomarkers of early hepatocarcinogenesis. [Copyright &y& Elsevier]

One of the most urgent requirements in nanotoxicology is a quantitative assessment of internalized nanomaterials in cells. We present an in vitro assay called 'max-flat' for the measurement of internalized particles in macrophages. Fluorescent polystyrene (PS) beads of diameters 1 µm, 500 nm, 200 nm, 100 nm and 20 nm were employed. Different concentrations of fibronectin (FN) coated substrates were tested to achieve a maximal cell spreading area and minimal nucleus height, hence 'max-flat'. We found this cell spreading area depends on FN concentration, and it is independent of particle concentration. An optimal condition of FN was found at 2.5×10-3 mg/ml, and it was selected for the max-flat assay to assess the internalization of PS beads. Cells under these conditions neither generate reactive oxygen species nor show noticeable differences in pro-survival/pro-apoptotic signals. Confocal images were employed for the max-flat assay and we set the interval scanning for a Z-stack as nucleus height divided by particle diameter. The max-flat assay provided a significantly higher number of internalized particles and the saturation is reached faster for nano-scale PS beads. We show how the proposed max-flat assay clearly outperform existing techniques by providing easier, more precise and far more reliable access to the number of internalized nanoparticles in macrophages. [ABSTRACT FROM AUTHOR]

Since the identification in the 1950s of deoxyribonucleic acid as the building block of life, the impact of molecular biology has been far-reaching. Understanding the processes of how DNA is replicated, transcribed into RNA and then translated into protein products has not only provided a fundamental knowledge of life but has also spawned a plethora of applications. Molecular biology has been high profile and widespread in research into the biology of disease and in drug discovery. It has additionally found application in understanding the adverse effects, or toxicity, of candidate drugs and how they interfere with biochemical and biological processes. In recent times the biggest impact of molecular biology in toxicology has been through the study of differential gene expression, largely as a result of the advent of genomics. This review seeks to describe how toxicogenomics strategies have been implemented and integrated into nonclinical studies of drug safety. [ABSTRACT FROM AUTHOR]

Abstract: Technologies that generate information about the genome are being used to explore changes in gene expression and related proteins following exposure to chemicals. Conceptually, this information allows a greater understanding of genomic level mRNA expression (transcriptomics), cell and tissue protein expression (proteomics) and information about metabolite profiles (metabonomics). Having a greater understanding of this information alongside, empirical toxicological reference data provide for the continued evolution in our ability to understand toxicological modes of action, thereby providing a more scientific basis for extrapolation of toxicological information from animals to humans. Toxicogenomics also provides specific opportunities for improvements at different stages of the risk assessment process such as the development of new predictive models for identifying human health hazards and the identification of more precise molecular biomarkers of exposure. One possibility is that molecular fingerprinting in vivo or in vitro can be used to categorize chemicals and mixtures of chemicals into different mode of action groups. As there is indication that molecular signals differ at dose levels, it is hoped that toxicogenomic information can also contribute to the understanding and interpretation of effects seen with low dose exposure. Several gene polymorphisms have already been identified which play a role in the differing intra-species response to chemicals thus providing explanation for the observed differences in effects. It is therefore likely that a better understanding of genomic expression will enable a greater insight into the factors behind the observed variability in susceptibility to chemical exposure that can be seen in human populations. [Copyright &y& Elsevier]

In order to investigate the fundamental principles that influence the optimal selection of toxicity test methods for the evaluation of chemical hazards, it is useful to have a design model to explore possible alternative testing strategies. In general, our lack of detailed knowledge of the mechanisms of toxicity (including not only the early events in the interaction of chemicals with biological systems, but the sequence of events that lead to experimentally measurable toxicity) limits the development of realistic dynamic models of the toxicological process for individual chemicals. We report here the development of a theoretical model that includes two independent hypothetical mechanisms of toxicity. The mechanisms are designed to be qualitatively similar to known mechanisms of action. The model is exercised to simulate the experimental data that would be obtained for a collection of “test” and “validation” chemicals using a single or a combination of two toxicity tests. The data generated are used to evaluate the “relevance” of the testing strategy based on the two proposed in vitro toxicity tests. [Copyright &y& Elsevier]