Showing papers in "Toxicology Research in 2015"

Interaction of tannic acid with carbon nanotubes: enhancement of dispersibility and biocompatibility

Abstract: The interaction of manufactured nanomaterials with environmental and biological systems has been a subject of great research interest for a long time. In the present study, adsorption of a universal environmental organic material (named tannic acid (TA)) on carbon nanotubes (CNTs) was investigated. The influence of CNT properties and pH values on the sorption capacity of CNTs for TA was also evaluated. Our results demonstrated that the sorption capacity of CNTs was positively correlated with their specific surface areas. Furthermore, TA could effectively enhance the water dispersibility of CNTs and reduce their cytotoxicity. Our results implied that TA could influence the environmental behavior and biological responses of the manufactured nanomaterials, reminding us that much more attention should be paid to the synergistic toxicity of nanomaterials when we evaluate their environmental impacts.

Silver nanoparticles – wolves in sheep's clothing?

Abstract: Silver nanoparticles (Ag NPs) are one of the most widely utilized engineered nanomaterials (ENMs) in commercial products due to their effective antibacterial activity, high electrical conductivity, and optical properties. Therefore, they have been one of the most intensively investigated nanomaterials in terms of their toxic effects on humans and the environment. It has become clear during recent years that nanomaterials can behave unexpectedly due to new and unique characteristics when their particle size reaches the nanoscale (1–100 nm). Consequently, their effect on human health and the environment has been hard to predict. Widespread applications increase the chances of public and environmental exposure to Ag NPs and have thereby increased concerns regarding the potential adverse effects of Ag NPs on human health and environmental safety. To fully understand and predict possible health effects following exposure to Ag NPs, information about the mechanisms for their cytotoxicity and genotoxicity is necessary. The present paper attempts to review the cellular and molecular mechanisms behind Ag NP toxicity. In addition, the role of silver ions in the toxicity of Ag NPs is discussed.

An unexpected increase of toxicity of amino acid-containing ionic liquids

Abstract: Functionalization of ionic liquids (ILs) with natural amino acids is usually considered as a convenient approach to decrease their toxicity and find new areas of chemical application as sustainable solvents, reagents or catalysts. In the present study, the cytotoxicity of several amino acid-containing ionic liquids (AAILs) with amino acid-based cations and anions was studied towards NIH/3T3 and CaCo-2 cell cultures and compared with the toxicity of conventional imidazolium-based ILs. The presence of an amino acid in the anion did not lead to a significant decrease in toxicity, whereas in the cation it unexpectedly increased the toxicity, as compared with conventional ILs. Exposure to 1-butyl-3-methylimidazolium chloride or 1-butyl-3-methylimidazolium glycinate induced apoptosis in NIH/3T3 cells. The present study gives new insights into biological effects of AAILs and shows that an amino acid residue may make ILs more biologically active. Special attention should be paid to the plausible synergetic effect of a combination of ILs with natural biologically active molecules. The results suggest possible medical application of AAILs rather than involvement as a green and sustainable tool to carry out chemical reactions.

The cytotoxic mechanisms of disulfiram and copper(II) in cancer cells

Abstract: The anticancer activity of disulfiram (DS) is copper(II) (Cu)-dependent. This study investigated the anticancer mechanisms of DS/Cu using in vitro cytotoxicity and metabolic kinetic analysis. Our study indicates that DS/Cu targets cancer cells by the combination of two types of actions: (1) instant killing executed by DS/Cu reaction generated reactive oxygen species; (2) delayed cytotoxicity introduced by the end product, DDC-Cu. Nanoencapsulation of DS might shed light on repositioning of DS into cancer treatment.

Manganese-induced Neurotoxicity: From C. elegans to Humans.

Abstract: Manganese (Mn) is one of the most abundant metals on the earth. It is required for normal cellular activities, but overexposure leads to toxicity. Neurons are more susceptible to Mn-induced toxicity than other cells, and accumulation of Mn in the brain results in Manganism that presents with Parkinson's disease (PD)-like symptoms. In the last decade, a number of Mn transporters have been identified, which improves our understanding of Mn transport in and out of cells. However, the mechanism of Mn-induced neurotoxicity is only partially uncovered, with further research needed to explore the whole picture of Mn-induced toxicity. In this review, we will address recent progress in Mn-induced neurotoxicity from C. elegans to humans, and explore future directions that will help understand the mechanisms of its neurotoxicity.

Uptake of gold nanoparticles in primary human endothelial cells

Abstract: Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy, nano-scale 3D-imaging using focused ion beam/scanning electron microscopy (FIB/SEM), and single particle inductively coupled plasma-mass spectrometry (spICP-MS). HUVECs were cultured for 3 h or 24 h in medium with AuNPs in a concentration range of 1.25–10 μg ml−1. There was a concentration-dependent increase of AuNPs inside cells measured by flow cytometry, spICP-MS and 3D confocal microscopy. The latter also showed that AuNPs were located in the cytosol. This was supported by FIB/SEM, showing that AuNPs were located in membrane enclosures in the cytoplasm as single particles or agglomerates of 2–3 or more particles. Pre-treatment with chlorpromazine inhibited the AuNP-uptake in HUVECs, indicating that internalisation occurred mainly by clathrin-mediated endocytosis. Cell activation by exposure to tumour necrosis factor or lipopolysaccharide had a slight or no effect on the uptake of AuNPs, respectively. The AuNP exposure did not influence cell cytotoxicity, whereas the intracellular reactive oxygen species production was slightly increased. In conclusion, the uptake of AuNPs by endothelial cells can be addressed quantitatively by several methods with high throughput and/or high specificity. Uptake of AuNPs in HUVECs occurred mainly by clathrin-mediated endocytosis and trafficking to membrane enclosures in the form of single particles and agglomerates of 2–3 particles.

Toxicity mechanism of graphene oxide and nitrogen-doped graphene quantum dots in RBCs revealed by surface-enhanced infrared absorption spectroscopy

Abstract: The study of the toxic effects of nanoparticles on biological systems at the molecular level is critical in order to gain a greater understanding of the origin of nanotoxicity. Recently, numerous forms of graphene materials have been synthesized and extensively applied in biosensors and biomedicine, but their toxicity has not yet been studied to the same extend, in particular the toxicity mechanism. In this work, we systematically studied the toxic effects of two typical graphene forms, graphene oxide (GO) and nitrogen-doped graphene quantum dots (N-GQDs), on red blood cells (RBCs) by testing their hemolytic activity, observing the morphological changes and detecting the ATP content of RBCs after being exposed to the two nanomaterials. The toxicity mechanism was further revealed by investigating the structural changes of RBCs lipid by surface-enhanced infrared absorption spectroscopy using model membranes. A detailed analysis of the infrared spectra revealed that the adsorption of GO destroys the integrity of a membrane by extracting the lipid bilayer, resulting in hemolysis and aberrant forms. In contrast, N-GQDs just disturb the structure and conformation of the lipid, resulting in only aberrant cells. To date, this is the first experimental study which has revealed the toxicity mechanism of graphene materials in RBCs at the molecular level.

Ah receptor pathway intricacies; signaling through diverse protein partners and DNA-motifs

Abstract: The Ah receptor is a transcription factor that modulates gene expression via interactions with multiple protein partners; these are reviewed, including the novel NC-XRE pathway involving KLF6.

Transgenerational safety of nitrogen-doped graphene quantum dots and the underlying cellular mechanism in Caenorhabditis elegans

Abstract: Nitrogen-doped graphene quantum dots (N-GQDs) are of potential use for cellular imaging. We here employed the in vivo Caenorhabditis elegans assay system to investigate the transgenerational behavior of N-GQDs and the underlying cellular mechanism involved. Prolonged exposure to N-GQDs did not induce lethality, lifespan reduction, or change the functions of primary and secondary targeted organs in the wild-type nematode and in nematodes with mutations of the sod-2 or sod-3 genes that encode Mn-SODs. Moreover, no adverse effects were detected in progeny of N-GQD-exposed wild-type and mutant nematodes. N-GQDs were only distributed in the intestine of both wild-type and mutant nematodes. No N-GQD accumulation was observed in embryos and progeny of exposed nematodes. After N-GQD exposure, the normal biological state of the intestinal barrier and defecation behavior were maintained in the wild-type and mutant nematodes. We hypothesize that the physiological states of the intestinal barrier and defecation behavior may contribute greatly to the lack of translocation of N-GQDs into secondary targeted organs and progeny of exposed nematodes. Our data provide systematic in vivo evidence to indicate the transgenerational safety of N-GQDs and the underlying cellular mechanism.

The puzzle of toxicity of gold nanoparticles. The case-study of HeLa cells

Abstract: Because of the growing interest of gold nanoparticles in biomedical and biotechnological applications, their toxicity is becoming an increasingly important issue and, in the last few years, there has been continuously expanding research activity in this field. However, due to the intrinsic complexity of the problem, together with the lack in the standardization of the experimental procedures, there is to date a large scattering of the results that still prevents reaching a general consensus of the possible toxic effects of gold nanoparticles in biological systems of increasing complexity (cell membrane, cells, tissues, organs and human body). The strong need to systematize the data suggests employing an appropriate metric, as far as the particle concentration is concerned, that could help in comparing and organizing the available data in a more intelligible scenario. In this note, some recent literature data on the viability of HeLa cells exposed to differently functionalized gold nanoparticles have been analyzed on the basis of a metric based on the numerical particle concentration (number of particles per unit volume of cell culture) that, to a certain extent, takes into account both the size and the shape of nanoparticles. This analysis offers a much more intelligible behavior than the one based on metrics that considers mass concentration (molar concentration) or particle size. At least, in the particular case of HeLa cells, the analysis of the data shows that differently functionalized gold nanoparticles behave similarly and that the different surface coating of the different nanoparticles considered defines the range of particle concentration where toxic effects begin. This kind of analysis could furnish some albeit preliminary suggestions towards an appropriate method to study gold nanoparticle toxicity.

Quantum dots exposure alters both development and function of D-type GABAergic motor neurons in nematode Caenorhabditis elegans

Abstract: We examined the in vivo quantum dots (QDs) neurotoxicity and the underlying mechanism using Caenorhabditis elegans D-type GABAergic motor neurons as the assay system. Prolonged exposure to low concentrations of CdTe QDs caused damage on both the development and function of D-type motor neurons, and resulted in translocation of CdTe QDs into D-type motor neurons. In addition to oxidative stress, cell identity was also involved in the induction of the toxicity of QDs on D-type motor neurons. ZnS surface coating blocked CdTe QDs translocation and maintained cell identity, thereby suppressing CdTe QDs neurotoxicity. For the underlying mechanism, we hypothesized that both translocation into the targeted neurons and alterations in the development and function of those targeted neurons contribute to the induction of CdTe QDs neurotoxicity. Considering the conserved property of GABAergic neurons during evolution, our data will shed light on our understanding of the potential risks of QDs to the nervous systems of animals.

Eugenia uniflora leaves essential oil induces toxicity in Drosophila melanogaster: involvement of oxidative stress mechanisms

Abstract: Eugenia uniflora L. (Myrtaceae family), also known as “pitanga”, is a tree species widely used in popular medicine. Despite the well documented beneficial effects of the extracts and essential oils from this plant, little is known about its toxicity. We performed a phytochemical fingerprinting and evaluated the toxicity induced by the Eugenia uniflora leaves essential oil in a Drosophila melanogaster model. In order to understand the biochemical mechanisms involved in E. uniflora essential oil toxicity, changes in the Nrf2 signaling as well as the hallmarks of oxidative stress were measured. The exposure of adult flies to the essential oil via a fumigant method resulted in increased mortality and locomotor deficits. In parallel, an oxidative stress response signaling, evidenced by changes in ROS production, lipid peroxidation, alterations in the activity of antioxidant enzymes and expression of Nrf2 protein targets occurred. In the light of our findings, attention is drawn to the indiscriminate use of this plant for medicinal purposes. In addition, a potential bio-insecticidal activity of Eugenia uniflora volatile compounds is suggested, a fact that needs to be further explored.

Biochemical and behavioral deficits in the lobster cockroach Nauphoeta cinerea model of methylmercury exposure

Abstract: Methylmercury (MeHg) is well-known for its neurodevelopmental effects both in animals and in humans. As an alternative to utilizing conventional animal models, this study evaluated behavioral and biochemical parameters using the nymphs of the lobster cockroach Nauphoeta cinerea. Animals were exposed to MeHg at 0, 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg per g feed for 35 consecutive days. Locomotor activity and exploratory profiles were analyzed using video-tracking software during a 10 minute trial. Subsequently, biochemical estimations were carried out using cockroach heads. MeHg exposure caused behavioral impairment as evidenced by a significant decrease in distance travelled, time spent walking, turn angle and body rotation. The marked decrease in the exploratory profiles of MeHg-exposed cockroaches was confirmed by track plots, whereas occupancy plot analyses revealed a gradual dispersal in home-base formation, starting from 0.0625 mg per g feed. Biochemically, MeHg exposure significantly decreased acetylcholinesterase activity (AChE), an enzyme which plays a pivotal role in neurotransmission. Moreover, MeHg caused increased oxidative stress as evidenced by decreased total thiol levels and glutathione S-transferase (GST) activity, along with increased 2′,7′-dichlorofluorescein (DFCH) oxidation and thiobarbituric acid reactive substance (TBARS) production. In conclusion, these data demonstrated that Nauphoeta cinerea mimics the behavioral and biochemical deficits observed in rodents exposed to MeHg, thus highlighting its validity as an alternative model for basic toxicological studies.

Development and use of in vitro alternatives to animal testing by the pharmaceutical industry 1980–2013

Abstract: We examined the use of in vitro (including in silico) techniques in preclinical safety testing by the pharmaceutical industry between 1980 and 2013 to determine patterns, drivers and challenges in uptake. Data were collected via a survey sent to the Association of the British Pharmaceutical Industry (ABPI) member companies from the Nonclinical and Biological Discovery Expert Network (NaBDEN) requesting the number of compounds screened using in vitro and in silico tests at 5-year intervals between 1980 and 2005 then yearly from 2008 onwards. A utility score from 1 (poor) to 5 (excellent) for each assay was also requested. Four pharmaceutical companies and 3 contract research organisations (CROs) responded to the survey, providing >895 000 data points across all years and all assays. Overall, there was a steady increase in the use of in vitro tests by the pharmaceutical industry between 1980 and 2013; indeed >20% of all in vitro tests reported were conducted in the last year of the survey window (2013) and >70% of all in vitro tests reported were conducted since 2010. Use of in vitro tests peaked at >190 000 tests per annum in 2012; >99% of this usage was in the three main areas reported of ADME, safety pharmacology and genotoxicity. Trends and step changes in uptake were most notable in the three main areas of ADME, safety pharmacology and genotoxicity and may be explained by the timing of adoption of the relevant International Committee on Harmonisation (ICH) guidelines. Trends in uptake may also be explained by perceptions of utility where scores varied from poor (Eye Irritation – flourescein leakage) to excellent (Genotoxicity – Ames and Skin irritation – EpiSkin/Epiderm). In summary, the data show a large increase and a continuing upwards trend in development and adoption of in vitro alternatives to animal testing in pharmaceutical drug development providing new opportunities to improve success rates coupled with a strong commitment to the 3Rs.

Effects of monoolein-based cubosome formulations on lipid droplets and mitochondria of HeLa cells

Abstract: Despite the remarkable development of nanoparticles for different purposes, relatively little is known about their interaction with biological systems and individual cells. Here the effects of two monoolein-based cubosome formulations stabilized by Pluronic F108 and F127 were investigated against HeLa cells. Microscopy analysis on living cells loaded with organelle-specific fluorescent probes was performed to assess the formation of cytoplasmic lipid droplets after nanoparticle treatment. Mitochondrial membrane potential and mitochondrial ROS generation were also investigated in relation to the capability of the accumulated lipids to affect mitochondrial functions. Values of the main cellular unsaturated fatty acids were also measured to assess cell lipid profile modulation. Results from this study show that the uptake of both cubosome formulations induced modification of the cell lipid profile, lipid droplet accumulation, mitochondrial hyperpolarization and mitochondrial ROS generation. These results shed some light on the influence exerted by monoolein-based cubosome formulations on subcellular organelles and their possible adverse effects on cell functions.

Biocompatibility of graphene oxide intravenously administrated in mice—effects of dose, size and exposure protocols

Abstract: Graphene oxide (GO) shows great promise in in vivo drug delivery and therapy applications. However, several reports have reported an in vivo toxicity of GO. In this study, we found that the toxicity of GO intravenously injected into mice could be tuned by dose, size and exposure protocols of GO. The exposure to a single dose of 2.1 mg kg−1 (single-high-dose exposure) small size GO or large size GO caused macrophage nodule formation in the lungs of the mice, and the exposure to seven repeated doses of 0.3 mg kg−1 (multiple-low-dose exposure) large size GO also induced small macrophage nodule formation, serious lymphocyte infiltration around the bronchioles in the lungs of the mice, and even death of the mice. Nephritic inflammatory reactions were also observed after the multiple-low-dose exposure to large size GO. However, no obvious lung toxicity but hepatic inflammatory infiltration was observed after the exposure to multiple-low-dose small size GO. GO accumulation in the macrophage nodules was verified by Raman mapping. These findings will benefit the applications of GO in the future, especially in biomedical fields.

Cellular response to the genotoxic insult: the question of threshold for genotoxic carcinogens

Abstract: Maintenance of cellular integrity is crucial for its physiological function, which is constantly threatened by DNA damage arising from numerous intrinsic and environmental sources. By transcribing the genetic information stored in the intact DNA, RNA polymerase II generates mRNA that instructs ribosomes to produce specific proteins. DNA damage leads to interruption of mRNA synthesis with the potential production of unstable transcriptions and proteins. DNA damage results in mutations with the possible consequence of cancer. To protect their integrity, eukaryotes including mammalian cells have developed different mechanisms including DNA damage response (DDR) to ensure protection of the genome. DDR identifies DNA lesions and, depending on the severity, triggers different responses. Mild DNA damage is normally managed by DNA repair; more severe or irreparable DNA damage triggers the induction of cell death programs such as apoptosis or necrosis. Although these defence mechanisms are increasingly being understood, the critical and rate limiting parameters and their dose–response to the insulting agent are poorly evaluated. From a toxicological point of view such information is essential to accept the existence of a threshold for genotoxic carcinogens. By describing the different cellular defence mechanisms and their regulation we intend to stress the need for such information to further evaluate the plausibility of a dose-dependent threshold mechanism of genotoxic carcinogens. Besides its scientific value, a better understanding of cellular defence and the onset of such counterbalancing reactions is of regulatory importance since a scientifically defendable threshold concept for genotoxic carcinogens will allow identification of the NOAEL and the derivation of health-based exposure limits.

In vitro toxicological characterisation of arsenic-containing fatty acids and three of their metabolites

Abstract: Arsenic-containing fatty acids are a group of fat-soluble arsenic species (arsenolipids) which are present in marine fish and other seafood. Recently, it has been shown that arsenic-containing hydrocarbons, another group of arsenolipids, exert toxicity in similar concentrations comparable to arsenite although the toxic modes of action differ. Hence, a risk assessment of arsenolipids is urgently needed. In this study the cellular toxicity of a saturated (AsFA 362) and an unsaturated (AsFA 388) arsenic-containing fatty acid and three of their proposed metabolites (DMAV, DMAPr and thio-DMAPr) were investigated in human liver cells (HepG2). Even though both arsenic-containing fatty acids were less toxic as compared to arsenic-containing hydrocarbons and arsenite, significant effects were observable at μM concentrations. DMAV causes effects in a similar concentration range and it could be seen that it is metabolised to its highly toxic thio analogue thio-DMAV in HepG2 cells. Nevertheless, DMAPr and thio-DMAPr did not exert any cytotoxicity. In summary, our data indicate that risks to human health related to the presence of arsenic-containing fatty acids in marine food cannot be excluded. This stresses the need for a full in vitro and in vivo toxicological characterisation of these arsenolipids.

In vitro models for neurotoxicology research

Abstract: The nervous system has a highly complex organization, including many cell types with multiple functions, with an intricate anatomy and unique structural and functional characteristics. The study of its (dys)functionality following exposure to xenobiotics, neurotoxicology, constitutes an important issue in neurosciences. Despite the extensive use of in vivo models to reveal the neurotoxicological phenomena, the existence of difficulties related to the increasing cost and time required for neurotoxicity studies with experimental animals, as well as the animal ethical concerns, have limited their use. Consequently, in vitro alternatives, providing an understanding of the mechanistic basis, at the molecular and cellular level, have earned a notable consideration in the field of neurotoxicological research. In this field, the selection of the most appropriate in vitro neuronal system relies on specific endpoints that are of particular relevance for the neurotoxicological phenomena that will be studied. Furthermore, application of specific endpoints to various neuronal cellular models should be done in a careful way to build reliable and feasible testing strategies. This review addresses the use of in vitro models for neurotoxicity research, aiming to contribute to a better understanding and guidance of in vitro neurotoxicological studies. As such, subcellular systems, namely isolated mitochondria and synaptosomes, and cellular models, including immortalized cell lines, primary cultures, co-cultures, organotypic cultures, neural stem cells and blood–brain barrier models, as well as their inherent advantages and limitations, are discussed.

Mitochondrial dysfunction and oxidative damage in the liver and kidney of rats following exposure to copper nanoparticles for five consecutive days

Abstract: Objective: The goal of the current study was to investigate the molecular mechanisms of copper nanoparticle (CuNP)-induced hepato- and nephrotoxicity by a proteomic analysis that was phenotypically anchored to conventional toxicological outcomes. Methods: We employed specialized proteomic techniques, namely two-dimensional difference gel electrophoresis coupled with mass spectrometry to analyze the changes in protein expression in rat liver and kidney after 5 days of oral copper nanoparticle administration. Serum biochemical analyses and histopathological examinations of livers and kidneys of all rats were also performed. Results: All of the results indicated that the adverse effects observed in the rats treated with 100 mg kg−1 d−1 nanocopper were less than those induced by 200 mg kg−1 d−1 CuNPs. Exposure to CuNPs at a dose of 200 mg kg−1 d−1 for 5 d can induce overt hepatotoxicity and nephrotoxicity through a mechanism that mainly involves scattered dot hepatocytic necrosis and widespread renal proximal tubule necrosis. In addition, significantly elevated copper accumulation, decreased thiol groups and elevated malondialdehyde levels were also observed in the liver and kidney tissues. The perturbed proteins identified in the rat livers and kidneys are mainly involved in the respiratory and energy metabolisms, antioxidant defense, phase II metabolism, lipid metabolism, urea cycle, creatine biosynthesis, intracellular calcium homeostasis, and cytoskeletal organization. No abnormalities were identified in the liver and kidney tissues from the rats treated with 200 mg kg−1 microcopper. Conclusions: The results of this study suggest that mitochondrial dysfunction and oxidative damage may be the initial events in the hepato- and nephrotoxicity of copper nanoparticles. The down-regulation of phase II metabolic enzymes in the liver and the decrease in calcium-binding proteins in the kidney appear to be specific modes of action in these target organs. Our findings offer new directions for future research aiming to identify the specific biomarkers of the hepatotoxicity and nephrotoxicity of copper nanoparticles.

Vitamin E ameliorates neurodegeneration related phenotypes caused by neurotoxicity of Al2O3-nanoparticles in C. elegans

Abstract: We investigated the protective effects of vitamin E against the in vivo neurotoxicity of Al2O3-nanoparticles (NPs) in Caenorhabditis elegans. Al2O3-NPs in the range of mg L−1 led to neurodegeneration related phenotypes including neuronal loss, abnormality of axon development, and gap formation on nerve cords in the GABAergic nervous system and some behavioral deficits. Pretreatment with 200 mg L−1 of vitamin E prevented the neurotoxicity of Al2O3-NPs by reducing both the neurodegeneration and behavioral deficits. As for the underlying mechanism, pretreatment with vitamin E prevented the induction of oxidative stress, and sustained the normal intestinal permeability and development in Al2O3-NPs exposed animals. Moreover, pretreatment with vitamin E inhibited the translocation of Al2O3-NPs through the intestinal barrier into other parts of nematodes. Vitamin E-pretreated animals also showed less abnormality in the development of neurons involved in behavioral control and expression pattern of genes regulating cell identity of the corresponding neurons. Our results will be helpful for designing effective strategies to ameliorate nanotoxicity.

Metabolomic profiling of emodin-induced cytotoxicity in human liver cells and mechanistic study

Abstract: Emodin is one of the most representative natural anthraquinone polyphenols and the liver is one of the major target organs for drug-induced toxicology. The hepatocyte is frequently affected due to its role in emodin metabolism and accumulation. Although the hepatotoxicity of emodin has been reported, its toxicological mechanism is still unclear. The purpose of the present study was to evaluate the cytotoxicity of emodin in cultured human normal liver cells (L-02), to investigate the toxicity-related metabolic pathways and to predict the possible toxicity mechanism. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytotoxicity tests demonstrated a concentration-dependent toxic effect of emodin on L-02 cells. Cells were treated for 48 h with low, medium and high doses of emodin, respectively, and then subjected to metabolomics analysis using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS). Intracellular metabolomics analysis revealed that emodin significantly disturbed cellular glutathione and fatty acid metabolism. In addition, an emodin–cysteine adduct was identified in cell culture medium, and its level increased with increasing concentrations of emodin. The possible relationship among metabolic disorders, adduct formation and emodin hepatotoxicity was also discussed. This study provides new insight into the cytotoxicity of emodin on metabolic pathways in human liver cells.

Biocompatibility studies on lanthanum oxide nanoparticles

Abstract: Lanthanum oxide nanoparticles (LONP), a rare earth metal oxide, have unique properties that make them a suitable candidate for several biomedical applications. We investigated certain key in vitro and in vivo biocompatibility endpoints on LONP. LONP were cytotoxic in in vitro assays and predominantly exerted their action via release of reactive oxygen species. These nanoparticles were neither irritants nor sensitizers in a rabbit model. LONP extracts did not exert any acute systemic toxicity effects in mice. On the other hand LONP exerted toxicity to the liver following oral administration, suggesting that these particles are absorbed from the gastrointestinal (GI) tract and deposited in the hepatobiliary system. LONP did not induce any mutation in the Ames test both in the presence or absence of S-9. These observations provide a base line biocompatibility and toxicity data on LONP. The current findings will also be useful in defining standards for nanoparticle containing devices.

Metabolomics reveals disturbed metabolic pathways in human lung epithelial cells exposed to airborne fine particulate matter

Abstract: Exposure to airborne fine particulate matter (PM2.5) has been associated with a variety of adverse health effects; however, the molecular mechanisms involved in PM2.5-elicited pulmonary toxicity are still not well elucidated. By conducting an ultra-high performance liquid chromatography/mass spectrometry-based metabolomics analysis, the present study investigated alterations of metabolome in human lung epithelial cells (A549) exposed to PM2.5 extracts. Distinct metabolomic profiles were found to be associated with PM2.5 treatment. PM2.5 significantly changed the abundance of 16 intracellular metabolites in a dose-dependent manner, of which 13 were decreased and three were increased. By pathway analysis, it was shown that the citrate cycle, amino acid biosynthesis and metabolism, and glutathione metabolism were the major metabolic pathways disturbed by PM2.5 in A549 cells. In addition, changes in expression of several key genes involved in these pathways further validated the metabolic alterations observed by metabolomics herein. It is suggested that PM2.5-induced oxidative stress may contribute to perturbation of metabolic processes occurring in cell mitochondria. Overall, these results aid in improving understanding of the toxicological mechanisms related to PM2.5, and identifying potential biomarkers indicative of inhalable PM2.5 exposure.

Toxicity evaluation and translocation of carboxyl functionalized graphene in Caenorhabditis elegans

Abstract: Carboxyl functionalized graphene (G-COOH) can be potentially used for biosensing and medical applications. However, little is known about the in vivo behavior and toxicity of G-COOH. To investigate the in vivo translocation and toxicity of G-COOH and the underlying cellular mechanism, we employed Caenorhabditis elegans as a model for toxicological study. Prolonged exposure to 0.01–100 mg L−1 of G-COOH from L1-larvae to adult day-1 did not cause any adverse effects on the lifespan, development, or functions of the intestine, neurons, and reproductive organs in exposed nematodes and their progeny. After prolonged exposure, G-COOH was not translocated into the secondary targeted organs such as reproductive organs and neurons or the body of the progeny of exposed nematodes. In the intestinal cells, G-COOH was mainly deposited in small-vesicle structures such as peroxisomes and lysosomes adjacent to microvilli and moderately deposited in the cytosol. Meanwhile, G-COOH exposed nematodes showed normal development and function of the intestine and normal biological function of the intestinal barrier. Moreover, G-COOH exposed nematodes had normal defection behavior and developmental state of AVL and DVB neurons that control the defecation behavior. Therefore, the G-COOH translocation pattern and functional state of the intestinal barrier and/or the defecation state may contribute greatly to the in vivo behavior and toxicity of G-COOH at concentrations less than 100 mg L−1 in nematodes. Our results provide useful information on the in vivo properties of G-COOH and its future applications.

Umbelliferone β-d-galactopyranoside exerts an anti-inflammatory effect by attenuating COX-1 and COX-2

Abstract: Umbelliferone β-D-galactopyranoside (UFG) (benzopyrone) is a member of the coumarin family, found in many plants exhibiting numerous pharmacological actions. The current experiments were carried out to exemplify the anti-inflammatory potential of UFG on chronic inflammation induced by Complete Freund Adjuvant (CFA) (heat killed Mycobacterium tuberculosis) in experimental rats. Arthritis in rats was induced by intradermal administration of CFA (0.05 ml) in the right hind paw, and confirmed by development of paw edema and arthritic index in comparison with normal controls. The anti-arthritic activity of UFG was determined by its ability to inhibit various biochemical markers, viz., pro-inflammatory, antioxidant enzymes, and hematological parameters elevated upon administration of CFA. UFG was also tested for its inhibitory activity against cyclooxygenase-1 (COX-1) and COX-2 via enzyme inhibition assay and the results were monitored spectrophotometrically with a 96-well plate reader. The results of the study showed that UFG in a dose of 10, 20 or 40 mg kg−1 per day, p.o., helps to prevent paw edema and arthritic score development for arthritis in rats. It markedly alters hematological and oxidative stress induced by the adjuvant. Moreover, the changes brought about in inflammation/arthritis serum markers were reverted back to a near normal level upon UFG treatment in a dose dependent manner. Histopathological analysis of the joints of subjects showed UFG significantly decreases mononuclear infiltration and synovial hyperplasia, which confirms the utility of UFG as an anti-arthritic agent. In a COX inhibition assay UFG was found to act prominently to inhibit COX-2 then COX-1, which suggests its plausible mechanism of action. The current study clearly indicates that UFG possesses anti-inflammatory effects against CFA induced arthritis via suppressing COX-2 inhibition.

Neurobehavioral changes induced by di(2-ethylhexyl) phthalate and the protective effects of vitamin E in Kunming mice

Abstract: Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer commonly used in PVC that may leach into the environment, and has been shown to adversely affect the health of humans and animals. We undertook a study to ascertain the neurotoxicity of DEHP in Kunming mice. This study included three rounds of testing. In the first round, Kunming mice were exposed to different concentrations of DEHP (0, 5, 50, 500 mg kg−1 per day) after which their cognitive ability was assessed using the Morris water maze (MWM) test. The reactive oxygen species (ROS) content in tissue and the malondialdehyde (MDA) content of brains were also measured. In the second round, vitamin E (50 mg kg−1 per day) was given daily as an anti-oxidant via the intragastric route. Cognitive deficits and locomotor activity, as well as ROS and MDA contents were tested employing the same methods. In the third round, the depressive mood of mice after DEHP exposure (500 mg kg−1 per day) was measured using the open field test, the tail suspension test, and the forced swim test. The main findings of this study include: (1) a statistical association exists between DEHP oral exposure and spatial learning (DEHP 500 mg kg−1 per day) and memory (DEHP 50 mg kg−1 per day) dysfunction as ascertained by an MWM test of Kunming mice. (2) A statistical association was also found between DEHP oral exposure (50 and 500 mg kg−1 per day) and oxidative stress (ROS and MDA) of mouse brain tissue. (3) Co-administration of vitamin E (50 mg kg−1 per day) diminishes the elevation of ROS and MDA induced by DEHP (50 mg kg−1 per day) from significant levels to non-significant levels. (4) Co-administration of vitamin E (50 mg kg−1 per day) protects against mouse memory dysfunction induced by DEHP (50 mg kg−1 per day) from being significant to being not significant. (5) In the 5 mg kg−1 per day DEHP exposure groups, oxidative stress in brain tissue, and neurobehavioral changes were not found. (6) High dose DEHP exposure (500 mg kg−1 per day) may induce behavioral despair in mice. Conclusions: These data suggest that DEHP is neurotoxic with regard to cognitive ability and locomotor activity.

Insulin signaling regulates the toxicity of traffic-related PM2.5 on intestinal development and function in nematode Caenorhabditis elegans

Abstract: Our previous study has demonstrated the adverse effects of traffic-related PM2.5 on Caenorhabditis elegans, a non-mammalian toxicological model. In this study, we employed the methods of genetics, gene expression pattern, intestinal autofluorescence, oxidative stress, and intestinal permeability to investigate the molecular control of in vivo toxicity from traffic-related PM2.5 by the insulin signaling pathway in nematodes. Among genes encoding the insulin signaling pathway, prolonged exposure to PM2.5 caused a decrease in the expression levels of daf-16 and daf-18 genes and an increase in the expression levels of daf-2, akt-1, and pdk-1 genes. Meanwhile, PM2.5 exposure increased the DAF-16::GFP nuclear localization. Mutations of the daf-2 gene encoding the insulin receptor restored deficits in both intestinal development and intestinal function in PM2.5 exposed nematodes. In contrast, mutations of the daf-16 gene encoding the FOXO transcription factor caused more severe damage on both intestinal development and intestinal function in PM2.5 exposed nematodes. Moreover, DAF-16 might regulate the toxicity of PM2.5 on the intestine through the functions of its downstream targets such as SOD-3, a manganese-superoxide dismutase. Therefore, the insulin signaling pathway may act as an important molecular basis for the toxicity of traffic-related PM2.5 in nematodes.

Euphorbia tirucalli aqueous extract induces cytotoxicity, genotoxicity and changes in antioxidant gene expression in human leukocytes

Abstract: Euphorbia tirucalli, popularly known as “avelos”, is a toxic plant used as tea in Brazilian folk medicine as an antibacterial, antiviral and anticarcinogenic agent. However, there is no scientific report about its potential toxicity in human cells. Therefore, the objective of the present study was to evaluate the in vitro genotoxicity and cytotoxicity of aqueous extracts of E. tirucalli in human leukocytes using a comet assay and trypan blue exclusion assay, respectively. In addition, the effect of E. tirucalli on the osmotic fragility was investigated in human erythrocytes. The expressions of selected antioxidant mRNA genes (SOD2, CAT and GPx4) as well as tumor protein p53 (TP53) were evaluated by qRT-PCR. Exposure of human leukocytes to high concentrations of aqueous extracts of E. tirucalli (100–150 μg mL−1) caused a significant increase in DNA damage. Leukocyte viability was decreased in the presence of 50–150 μg mL−1E. tirucalli extract. E. tirucalli did not change the osmotic fragility of human erythrocytes. High concentrations of E. tirucalli (10–50 μg mL−1) significantly up-regulated the mRNA of SOD2 and CAT and decreased the mRNA of GPx4 expression in human leukocytes. In addition, the mRNA gene expression of SOD2 was down-regulated at the highest concentration tested (150 μg mL−1). In summary, based on the results of genotoxicity observed in our study, we recommend caution regarding the acute or chronic use of this homemade preparation. Taken together, our results suggest that the aqueous extract of E. tirucalli induces genotoxicity and cytotoxicity in human leukocytes, possibly by interacting with the antioxidant enzyme system, thereby, increasing the formation of ROS and decreasing the cellular tolerance level to chemical constituents of this plant.

Beyond a means of exposure: a new view of the mother in toxicology research

Abstract: Toxicological studies generally view pregnant animals as a conduit through which gestational exposure of offspring to chemicals can be achieved, allowing for the study of developmental toxicity. One class of compounds, endocrine disrupting chemicals (EDCs), has been shown to interfere with hormone signalling in a number of in vivo models, with effects that are more pronounced when exposures occur during development. Yet, evidence has also accumulated that the traditional means of assessing the health effects of EDCs, using test guidelines, are not sufficient to fully capture the effects of these compounds. Our work has focused on the effects of EDCs on more complex, integrated endpoints than the ones typically examined in test guidelines (such as organ weight, histopathology, number of live/dead offspring). Here, we review evidence that EDCs interfere with the display of maternal behaviours, integrated endpoints affected by multiple hormones and other signalling molecules. We discuss a number of assays that can be easily conducted to assess the effects of environmental chemicals on maternal behaviours, as well as molecular assays of the medial preoptic area (MPOA), considered one of the principal regions in the brain responsible for maternal responsiveness. We propose that studies of emerging EDCs of concern should examine maternal behaviours in developmental toxicity tests, and when effects are observed, the maternal brain should be examined as well. We further advise that researchers avoid viewing the mother as simply a means of exposing the group of interest (i.e. the offspring), and instead recognize how the mother can greatly influence the behavioural phenotype and health of her offspring.