The potential health challenges of TiO2 nanomaterials

Titanium dioxide (TiO₂) nanomaterials (NMs) have found widespread applications owing to their attractive physical and chemical properties. As a result, the potential adverse impacts of nano‐TiO₂ exposure on humans have become a matter of concern. This review presents the state‐of‐the‐art advances on the investigations of the adverse effects of NMs, including the potential exposure routes of nano‐TiO₂ (e.g. respiratory system, skin absorption and digestive system), the physico‐chemical characterizations of nano‐TiO₂ (e.g. crystal structure, shape,size, zeta potential, treatment media, aggregation and agglomeration tendency, surface characteristics and coatings), risk evaluation of nanotoxicity (e.g. cytotoxicity, ecotoxicity, phototoxicity, and phytotoxicity) and potential mechanisms of adverse effects (e.g. generation of reactive oxygen species, oxidative stress and organelle dysfunction). The review aims to facilitate scientific assessments of health risks to nano‐TiO₂, which would guide the safe applications of NMs in our daily life. Copyright © 2015 John Wiley & Sons, Ltd.     

Nano-titanium dioxide induced cardiac injury in rat under oxidative stress

Heart diseases, which are related to oxidative stress (OS), negatively affect millions of people from kids to the elderly. Titanium dioxide (TiO₂) has widespread applications in our daily life, especially nanoscale TiO₂. Compared to the high risk of particulate matter (⩽2.5 μm) in air to heart disease patients, related research of TiO₂ on diseased body is still unknown, which suggest us to explore the potential effects of nanoscale and microscale TiO₂ to heart under OS conditions. Here, we used alloxan to induce OS conditions in rat, and investigated the response of heart tissue to TiO₂ in healthy and alloxan treated rats. Compared with NMs treatment only, the synergistic interaction between OS conditions and nano-TiO₂ significantly reduced the heart-related function indexes, inducing pathological changes of myocardium with significantly increased levels of cardiac troponin I and creatine kinase-MB. In contrast with the void response of micro-TiO₂ to heart functions in alloxan treated rats, aggravation of OS conditions might play an important role in cardiac injury after alloxan and nano-TiO₂ dual exposure. Our results demonstrated that OS conditions enhanced the adverse effects of nano-TiO₂ to heart, suggesting that the use of NMs in stressed conditions (e.g., drug delivery) needs to be carefully monitored.     

An in vivo study of nanorod,nanosphere, and nanowire forms of titanium dioxide using Drosophila melanogaster: toxicity,cellular uptake,oxidative stress,and DNA damage

ABSTRACT

The biological impact of nanomaterials (NMs) is determined by several factors such as size and shape, which need to be taken into consideration in any type of analysis. While investigators often prefer to conduct in vitro studies for detection of any possible adverse effects of NMs, in vivo approaches yield more relevant data for risk assessment. For this reason, Drosophila melanogaster was selected as a suitable in vivo model to characterize the potential risks associated with exposure nanorods (NRs), nanospheres (NSs), nanowires (NWs) forms of titanium dioxide (TiO₂), and their microparticulated (or bulk) form, as TiO₂. Third instar larvae (72 hr old larvae) were fed with TiO₂ (NRs, NSs, or NWs) and TiO₂ at concentrations ranging from 0.01 to 10 mM. Viability (toxicity), internalization (cellular uptake), intracellular reactive oxygen species (ROS) production, and genotoxicity (Comet assay) were the end-points evaluated in hemocyte D. melanogaster larvae. Significant intracellular oxidative stress and genotoxicity were noted at the highest exposure concentration (10 mM) of TiO₂ (NRs, NSs, or NWs), as determined by the Comet assay and ROS analysis, respectively. A concentration–effect relationship was observed in hemocytes exposed to the NMs. Data demonstrated that selected forms of TiO₂.-induced genotoxicity in D. melanogaster larvae hemocytes indicating this organism is susceptible for use as a model to examine in vivo NMs-mediated effects.     

Minireview: does in‐vitro testing of oximes help predict their in‐vivo action after paraoxon exposure?

K‐oximes have recently been developed in the search for efficacious broad‐band reactivators of acetylcholinesterase (AChE) inhibited by organophosphorus compounds (OPC). Before clinical use, their toxicity and efficacy need to be assessed, and there is clear demand for simple in vitro tests that can predict in vivo performance. This article summarizes our in vitro data obtained for conventional and experimental oximes in human and rat blood exposed to the OPC paraoxon and correlates them with our in vivo results. The intrinsic AChE inhibitory activity of oximes, as reflected by their in vitro IC₅₀, is strongly correlated with their LD₅₀ (rat): oximes with a high IC₅₀ (K‐27, K‐48, pralidoxime and obidoxime) also show a high LD₅₀ and are thus relatively non‐toxic, whereas oximes K‐105, K‐108 and K‐113 have a low IC₅₀, a low LD₅₀ and are far more toxic. The IC₅₀ is also correlated with the in vivo capacity to protect from paraoxon‐induced mortality: oximes with a higher IC₅₀ reduce the relative risk of death more. In contrast, the protective ability as assessed in vitro by the slope of the IC₅₀ shift (tanα), is not correlated with in vivo protection from paraoxon‐induced mortality: the best in vivo protectors (K‐27 and K‐48) show a much lower tanα value (around 2) than K‐110 and K‐113 (tanα around 10), which hardly reduce the relative risk of death after paraoxon exposure. The partition coefficient logP of the individual oximes is inversely correlated with their IC₅₀ and with their LD₅₀ and is therefore an indicator of toxicity: strongly hydrophilic oximes tend to be less toxic than less hydrophilic ones. These data highlight the good predictive value of in vitro IC₅₀ testing for in vivo toxicity and the limited practical significance of in vitro assessment of protective potency. Copyright © 2009 John Wiley & Sons, Ltd.     

Nanomaterial-induced cell death in pulmonary and hepatic cells following exposure to three different metallic materials: The role of autophagy and apoptosis

Autophagy is the catabolic process involving the sequestration of the cytoplasm within double-membrane vesicles, which fuse with lysosomes to form autolysosomes in which autophagic targets are degraded. Since most endocytic routes of nanomaterial uptake converge upon the lysosome and the possibility that autophagy induction by NMs may be an attempt by the cell to self-preserve following the external challenge, this study investigated the role of autophagy following exposure to a panel of widely used metal-based NMs with high toxicity (Ag and ZnO) or low toxicity (TiO₂) in a pulmonary (A549) and hepatic (HepG2) cell line. The in vitro exposure to the Ag and ZnO NMs resulted in the induction of both apoptosis and autophagy pathways in both cell types. However, the progression of autophagy was blocked in the formation of the autolysosome, which coincided with morphologic changes in the actin cytoskeleton. This response was not observed following the exposure to low-toxicity TiO₂ NMs. Overall, the results show that high toxicity NMs can cause a dysfunction in the autophagy pathway which is associated with apoptotic cell death.     

Endothelial cell activation,oxidative stress and inflammation induced by a panel of metal-based nanomaterials

Abstract

The importance of composition, size, crystal structure, charge and coating of metal-based nanomaterials (NMs) were evaluated in human umbilical vein endothelial cells (HUVECs) and/or THP-1 monocytic cells. Biomarkers of oxidative stress and inflammation were assessed because they are important in the development of cardiovascular diseases. The NMs used were five TiO₂ NMs with different charge, size and crystal structure, coated and uncoated ZnO NMs and Ag which were tested in a wide concentration range. There were major differences between the types of NMs; exposure to ZnO and Ag resulted in cytotoxicity and increased gene expression levels of HMOX1 and IL8. The intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) expression were highest in TiO₂ NM-exposed cells. There was increased adhesion of THP-1 monocytic cells onto HUVECs with Ag exposure. None of the NMs increased the intracellular ROS production. There were no major effects of the coating of ZnO NMs. The TiO₂ NMs data on ICAM-1 and VCAM-1 expression suggested that the anatase form was more potent than the rutile form. In addition, the larger TiO₂ NM was more potent than the smaller for gene expression and ICAM-1 and VCAM-1 expression. The toxicological profile of cardiovascular disease-relevant biomarkers depended on composition, size and crystal structure of TiO₂ NMs, whereas the charge on TiO₂ NMs and the coating of ZnO NMs were not associated with differences in toxicological profile.     

Synthesis,molecular docking,and pharmacological evaluation of N‐(2‐(3,5‐dimethoxyphenyl)benzoxazole‐5‐yl)benzamide derivatives as selective COX‐2 inhibitors and anti‐inflammatory agents

A series of N‐(2‐(3,5‐dimethoxyphenyl)benzoxazole‐5‐yl)benzamide derivatives ( 3am ) was synthesized and evaluated for their in vitro inhibitory activity against COX‐1 and COX‐2. The compounds with considerable in vitro activity (IC₅₀ < 1 μM) were evaluated in vivo for their anti‐inflammatory potential by the carrageenan‐induced rat paw edema method. Out of 13 newly synthesized compounds, 3a , 3b , 3d , 3g , 3j , and 3k were found to be the most potent COX‐2 inhibitors in the in vitro enzymatic assay, with IC₅₀ values in the range of 0.06–0.71 μM. The in vivo anti‐inflammatory activity of these six compounds ( 3a , 3b , 3d , 3g , 3j , and 3k ) was assessed by the carrageenan‐induced rat paw edema method. Compounds 3d (84.09%), 3g (79.54%), and 3a (70.45%) demonstrated significant anti‐inflammatory activity compared to the standard drug ibuprofen (65.90%) and were also found to be safer than ibuprofen, by ulcerogenic studies. A docking study was done using the crystal structure of human COX‐2, to understand the binding mechanism of these inhibitors to the active site of COX‐2.

     

Comparison of toxicity of different nanorod‐type TiO2 polymorphs in vivo and in vitro

It is predicted that the toxicity of nanoparticles may be different depending on the properties of the nanoparticles and biological system being tested. However, the factors that influence the toxicity of nanoparticles have not been adequately investigated. In this study, we characterized two types of TiO₂ nanorods, anatase (ATO) and brookite (BTO), and compared their toxicity in vivo and in vitro. ATO and BTO differed from each other most notably in their surface areas. Treatment with the two TiO₂ nanorods (10 µg ml^–1) produced similar effects on the cell cycle in eight cell lines which are derived from potential target organs of nanoparticles, with the BTO eliciting stronger responses than ATO in all cell lines, among the cell lines, H9C2 showed the maximal change. Similarly, when mice were exposed to two TiO₂ nanorods (1 mg kg^–1), BTO induced clearer histopathological lesions and triggered a more robust secretion of inflammatory cytokines than ATO. Furthermore, we compared the cellular response of both TiO₂ nanorods using BEAS‐2B cells, the human bronchial epithelial cell line. Both nanorods induced cell death by increasing the formation of autophagosome‐like vacuoles. The mitochondrial calcium concentration decreased by exposure of both types, but the distribution of lysosome and endoplasmic reticulum (ER) showed a clear difference between the two nanorods. Thus, we conclude that the surface area acts as an important factor which depends on toxicity of nanorod type‐TiO₂ nanoparticles. Furthermore, the toxicity of nanoparticles varies according to the type of cells tested, and that the assembly of autophagosome‐like vacuoles is a critical part of the cellular response to nanoparticle exposure. Copyright © 2013 John Wiley & Sons, Ltd.     

In‐vitro and in‐vivo antivenin activity of 2‐[2‐(5,5,8a‐trimethyl‐2‐methylene‐decahydro‐naphthalen‐1‐yl)‐ethylidene]‐succinaldehyde against Ophiophagus hannah venom

Objectives Curcuma zedoaroides A. Chaveerach & T. Tanee, locally known as Wan‐Paya‐Ngoo‐Tua‐Mia, is commonly used in the North‐Eastern part of Thailand as a ‘snakebite antidote’. The aim of this study was to isolate the active compound from the rhizome of C. zedoaroides, to determine its structure and to assess its antagonistic activity in vitro and in vivo against King cobra venom. Methods The active compound was obtained from C. zedoaroides by extraction with acetone followed by purification using column chromatography; its X‐ray structure was determined. Its inhibition of venom lethality was studied in vitro in rat phrenic nerve‐hemidiaphragms and in vivo in mice. Key findings The acetone extract of the Curcuma rhizomes contained a C20 dialdehyde, [2‐(5,5,8a‐trimethyl‐2‐methylene‐decahydro‐naphthalen‐1‐yl)‐ethylidene]‐succinaldehyde, as the major component. The isolated curcuma dialdehyde was found active in vitro and in vivo for antivenin activity against the King cobra venom. Using isolated rat phrenic nerve‐hemidiaphragm preparations, a significant antagonistic effect on the inhibition of neuromuscular transmission was observed in vitro. Inhibition on muscle contraction, produced by the 4 μg/ml venom, was reversed by 2–16 μg/ml of Curcuma dialdehyde in organ bath preparations over a period of 2 h. Mice intraperitoneally injected with 0.75 mg/kg venom and dialdehyde at 100 mg/kg had a significantly increased survival time. Injection of Curcuma dialdehyde (100 mg/kg) 30 min before the subcutaneous injection of the venom resulted in a 100% survival time after 2 h compared with 0% for the control group. Conclusions The in vitro and in vivo evaluation confirmed the medicinal use of traditional snake plants against snakebites. The bioactivity is linked to an isolated molecule and not a result of synergistic effects of a mixture. The active compound was isolated and the structure fully elucidated, including its stereochemistry. This dialdehyde is a versatile chemical building block and can be easily obtained from this plant source.     

Molecular mechanism of nanoparticulate TiO2 induction of axonal development inhibition in rat primary cultured hippocampal neurons

Numerous studies have demonstrated the in vitro and in vivo neurotoxicity of nanoparticulate titanium dioxide (nano‐TiO₂), a mass‐produced material for a large number of commercial and industrial applications. The mechanism of nano‐TiO₂‐induced inhibition of axonal development, however, is still unclear. In our study, primary cultured hippocampal neurons of 24‐hour‐old fetal Sprague‐Dawley rats were exposed to 5, 15, or 30 μg/mL nano‐TiO₂ for 6, 12, and 24 hours, and the toxic effects of nano‐TiO₂ exposure on the axons development were detected and its molecular mechanism investigated. Nano‐TiO₂ accumulated in hippocampal neurons and inhibited the development of axons as nano‐TiO₂ concentrations increased. Increasing time in culture resulted in decreasing axon length by 32.5%, 36.6%, and 53.8% at 6 hours, by 49.4%, 53.8%, and 69.5% at 12 hours, and by 44.5%, 58.2%, and 63.6% at 24 hours, for 5, 15, and 30 μg/mL nano‐TiO₂, respectively. Furthermore, nano‐TiO₂ downregulated expression of Netrin‐1, growth‐associated protein‐43, and Neuropilin‐1, and promoted an increase of semaphorin type 3A and Nogo‐A. These studies suggest that nano‐TiO₂ inhibited axonal development in rat primary cultured hippocampal neurons and this phenomenon is related to changes in the expression of axon growth‐related factors.     

Fibrinogen enhances the inflammatory response of alveolar macrophages to TiO2, SiO2 and carbon nanomaterials

Many studies have shown that the composition of the protein corona dramatically affects the response of cells to nanomaterials (NMs). However, the role of each single protein is still largely unknown. Fibrinogen (FG), one of the most abundant plasma proteins, is believed to mediate foreign-body reactions. Since this protein is absent in cell media used in in vitro toxicological tests the possible FG-mediated effects have not yet been assessed. Here, the effect of FG on the toxicity of three different kinds of inorganic NMs (carbon, SiO₂ and TiO₂) on alveolar macrophages has been investigated. A set of integrated techniques (UV–vis spectroscopy, dynamic light scattering and sodium dodecyl sulphate-polyacrylamide gel electrophoresis) have been used to study the strength and the kinetics of interaction of FG with the NMs. The inflammatory response of alveolar macrophages (MH-S) exposed to the three NMs associated with FG has also been investigated. We found that FG significantly enhances the cytotoxicity (lactate dehydrogenase leakage) and the inflammatory response (increase in nitric oxide (NO) concentration and NO synthase activation) induced by SiO₂, carbon and TiO₂ NMs on alveolar macrophages. This effect appears related to the amount of FG interacting with the NMs. In the case of carbon NMs, the activation of fibrinolysis, likely related to the exposure of cryptic sites of FG, was also observed after 24?h. These findings underline the critical role played by FG in the toxic response to NMs.     

Multilaboratory evaluation of 15 bioassays for (eco)toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID

Within EU FP7 project NANOVALID, the (eco)toxicity of 7 well-characterized engineered nanomaterials (NMs) was evaluated by 15 bioassays in 4 laboratories. The highest tested nominal concentration of NMs was 100?mg/l. The panel of the bioassays yielded the following toxicity order: Ag?>?ZnO?>?CuO?>?TiO_2?>_?MWCNTs?>?SiO_2?>_?Au. Ag, ZnO and CuO proved very toxic in the majority of assays, assumingly due to dissolution. The latter was supported by the parallel analysis of the toxicity of respective soluble metal salts. The most sensitive tests/species were Daphnia magna (towards Ag NMs, 24-h EC_50?=_?0.003?mg Ag/l), algae Raphidocelis subcapitata (ZnO and CuO, 72-h EC_50?=_?0.14?mg Zn/l and 0.7?mg Cu/l, respectively) and murine fibroblasts BALB/3T3 (CuO, 48-h EC_50?=_?0.7?mg Cu/l). MWCNTs showed toxicity only towards rat alveolar macrophages (EC_50?=_?15.3?mg/l) assumingly due to high aspect ratio and TiO₂ towards R. subcapitata (EC_50?=_?6.8?mg Ti/l) due to agglomeration of TiO₂ and entrapment of algal cells. Finally, we constructed a decision tree to select the bioassays for hazard ranking of NMs. For NM testing, we recommend a multitrophic suite of 4 in vitro (eco)toxicity assays: 48-h D. magna immobilization (OECD202), 72-h R. subcapitata growth inhibition (OECD201), 30-min Vibrio fischeri bioluminescence inhibition (ISO2010) and 48-h murine fibroblast BALB/3T3 neutral red uptake in vitro (OECD129) representing crustaceans, algae, bacteria and mammalian cells, respectively. Notably, our results showed that these assays, standardized for toxicity evaluation of “regular” chemicals, proved efficient also for shortlisting of hazardous NMs. Additional assays are recommended for immunotoxicity evaluation of high aspect ratio NMs (such as MWCNTs).     

Aryl hydrocarbon receptor‐mediated effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin on glucose‐stimulated insulin secretion in mice

Epidemiological and laboratory studies suggested that exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) affects glucose homeostasis and increases the incidence of type 2 diabetes mellitus. To evaluate the effects of TCDD on insulin secretion from islets of Langerhans (islets), we designed in vivo, ex vivo and in vitro experiments. For the in vivo experiment, male C57BL/6J and aryl hydrocarbon receptor (AhR)‐null mice were injected intraperitoneally with TCDD (10 μg kg^?1 b.w.), fasted for 12 h and administered glucose 24 h post‐administration. TCDD exposure significantly decreased the plasma insulin concentration at 60 and 120 min after a glucose challenge in C57BL/6J mice but not in AhR‐null mice. In contrast, the plasma glucose concentration was not changed by TCDD exposure in both C57BL/6J and AhR‐null mice. For the ex vivo experiment, we isolated islets 24 h after TCDD administration and determined the glucose‐stimulated insulin secretion from the islets. The insulin secretion level was found to be significantly decreased by TCDD exposure at 60 min after glucose treatment. For the in vitro experiment, islets harvested from untreated C57BL/6J mice were exposed to 0.1, 1, 10 or 100 nM TCDD for 24 h and analyzed for glucose‐stimulated insulin secretion. Insulin secretion from the islets remained unchanged regardless of TCDD dose. In conclusion, TCDD exposure impaired the second phase of glucose‐stimulated secretion of insulin from the islets via the AhR signaling pathway. Copyright © 2009 John Wiley & Sons, Ltd.     

Applicability of rat precision-cut lung slices in evaluating nanomaterial cytotoxicity,apoptosis, oxidative stress,and inflammation

The applicability of rat precision-cut lung slices (PCLuS) in detecting nanomaterial (NM) toxicity to the respiratory tract was investigated evaluating sixteen OECD reference NMs (TiO₂, ZnO, CeO₂, SiO₂, Ag, multi-walled carbon nanotubes (MWCNTs)). Upon 24-hour test substance exposure, the PCLuS system was able to detect early events of NM toxicity: total protein, reduction in mitochondrial activity, caspase-3/-7 activation, glutathione depletion/increase, cytokine induction, and histopathological evaluation. Ion shedding NMS (ZnO and Ag) induced severe tissue destruction detected by the loss of total protein. Two anatase TiO₂ NMs, CeO₂ NMs, and two MWCNT caused significant (determined by trend analysis) cytotoxicity in the WST-1 assay. At non-cytotoxic concentrations, different TiO₂ NMs and one MWCNT increased GSH levels, presumably a defense response to reactive oxygen species, and these substances further induced a variety of cytokines. One of the SiO₂ NMs increased caspase-3/-7 activities at non-cytotoxic levels, and one rutile TiO₂ only induced cytokines. Investigating these effects is, however, not sufficient to predict apical effects found in vivo. Reproducibility of test substance measurements was not fully satisfactory, especially in the GSH and cytokine assays. Effects were frequently observed in negative controls pointing to tissue slice vulnerability even though prepared and handled with utmost care. Comparisons of the effects observed in the PCLuS to in vivo effects reveal some concordances for the metal oxide NMs, but less so for the MWCNT. The highest effective dosages, however, exceeded those reported for rat short-term inhalation studies. To become applicable for NM testing, the PCLuS system requires test protocol optimization.     

Alpha‐phellandrene‐induced apoptosis in mice leukemia WEHI‐3 cells in vitro

Although reports have shown that α‐phellandrene (α‐PA) is one of the monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α‐PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α‐PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α‐PA on the cell morphology, viability, cell cycle distribution, and apoptosis in mice leukemia WEHI‐3 cells in vitro. Results indicated that α‐PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub‐G1 phase (apoptosis) in WEHI‐3 cells. α‐PA increased the productions of reactive oxygen species (ROS) and Ca²⁺ and decreased the levels of mitochondrial membrane potential (ΔΨ_m) in dose‐ and time‐dependent manners in WEHI‐3 cells that were analyzed by flow cytometer. Results from confocal laser microscopic system examinations show that α‐PA promoted the release of cytochrome c, AIF, and Endo G from mitochondria in WEHI‐3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α‐PA induces cell cycle arrest and apoptosis in WEHI‐3 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1640–1651, 2016.     

Serum enhanced cytokine responses of macrophages to silica and iron oxide particles and nanomaterials: a comparison of serum to lung lining fluid and albumin dispersions

The potential hazard to humans exposed to nanomaterials such as silica and iron oxide was investigated using an in vitro macrophage cell culture system. Amorphous silica and iron oxide particles and nanomaterials (NMs) were dispersed in cell culture medium supplemented with either bovine serum albumin (BSA), lung lining fluid (LLF) or serum, in order to mimic the body fluids encountered during different routes of exposure in the body. End points investigated included macrophage viability and cytokine production. Silica NMs and particles (50 and 200 nm, respectively) were unmodified (plain) or aminated (NH₂). Iron oxide NMs and particles, Fe₃O₄ 45 nm and Fe₂O₃ 280 nm were also used in this study. Silica particles and NMs induced a dose‐dependent increase in cytotoxicity as measured by lactate dehydrogenase (LDH) release. Serum enhanced silica‐induced interleukin (IL)‐6, IL‐10, IL‐1β and MCP‐1 release, whereas albumin partially inhibited MCP‐1 release. Aminated silica, 50 nm was more potent than the 200‐nm particles at inducing monocyte chemoattractant protein‐1 (MCP‐1) production when dispersed in medium or LLF, suggesting a size specific effect for these particles and this cytokine. Iron oxide particles were relatively inert compared with the silica particles and NMs; however, serum and albumin did affect cytokine release in some treatments. In conclusion, the data suggests that serum, compared with medium, BSA and LLF is very potent at enhancing macrophage responses to silica and iron oxide particles and NMs. Size was only influential in LLF for a limited number of parameters, whereas surface chemistry was not of consequence in this in vitro macrophage system. Copyright © 2014 John Wiley & Sons, Ltd.     

Cadmium inhibits the ovary δ‐aminolevulinate dehydratase activity in vitro and ex vivo: protective role of seleno‐furanoside

Cadmium (Cd) toxicity is a concern to the tobacco‐smoking sub‐population which includes millions of people worldwide. Although this metal may cause severe damage to embryos and the reproductive organs, the precise mechanisms underlying its toxicity remain unclear. In the present study, the Cd effect on ovary δ‐aminolevulinate dehydratase (δ‐ALA‐D) activity was investigated in vitro and ex vivo. We observed that low concentrations of Cd inhibited cow ovary δ‐ALA‐D activity in vitro and the IC₅₀ value obtained was 19.17 μM. Furthermore, the protective effect of a novel organic selenium compound (seleno‐furanoside) in restoring enzyme activity was evaluated. Seleno‐furanoside (10, 50, 100, 200, 400 and 1000 μM) did not reverse the Cd toxicity in bovine ovarian tissue in vitro. According to the in vitro reults, acute Cd exposure (2.5 and 5 mg kg^–1) caused a significant inhibition in ovary δ‐ALA‐D activity in mice (around 27% and 34%, respectively). Therapy with seleno‐furanoside (100 µmol kg^–1) was able to restore enzyme activity. Thus, we demonstrated for the first time that δ‐ALA‐D activity from ovary is inhibited by Cd both in vitro and ex vivo. Additionally, seleno‐furanoside therapy was effective in restoring ovarian enzyme activity inhibited by Cd exposure in mice, but it did not reverse the in vitro metal effect. This study detected a new toxicity marker of Cd toxicity on ovarian tissue as well as the beneficial effect of a new compound to manage the metal effect after acute exposure. Copyright © 2012 John Wiley & Sons, Ltd.     

Hepatotoxicity and liver injury induced by hydroxyapatite nanoparticles

As hydroxyapatite nanoparticles (HA NPs) are increasingly used in biomedical and biotechnological fields, risk assessment of HA NPs has attracted extensive attention. Nevertheless, little is known about the potential adverse effects of HA NPs on normal hepatocytes and the liver. In the present study, we conducted an in vitro study in which 80‐nm HA NPs were incubated with normal Buffalo rat liver (BRL) cells. By analyzing the changes in cell viability, apoptosis/necrosis and the mitogen‐activated protein kinase (MAPK) signaling pathway, we investigated the cytotoxicity and potential mechanism of HA NPs in hepatocytes. Furthermore, we used the serum hematology and histopathology examinations to explore the in vivo effects of HA NPs on the structure and function of the liver. Our results showed that exposure to HA NPs at a concentration above 200 µg ml^?1 decreased cell viability, increased levels of lactate dehydrogenase (LDH) leakage, induced apoptosis and necrosis, and triggered the MAPK signaling pathway in BRL cells in a dose‐dependent manner. Moreover, our in vivo study indicated that HA NPs increased the white blood cell count (WBC) and the levels of tumor necrosis factor‐α (TNF‐α), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum, caused inflammatory cell infiltration at the portal area in the liver, and induced hepatic oxidative stress with elevated levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). These data demonstrate that at certain concentrations, 80‐nm HA NPs cause hepatotoxicity and liver injury. Copyright © 2014 John Wiley & Sons, Ltd.     

The in vivo and in vitro phase I metabolism of FYL‐67, a novel oxazolidinone antibacterial drug,studied by LC‐MS/MS

In our previous study, FYL‐67, a novel linezolid analogue with the morpholinyl ring replaced by a 4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl group, was demonstrated to own an excellent activity against Gram‐positive organisms,such as methicillin‐resistant Staphylococcus aureus (MRSA). However, metabolic biotransformation was not investigated. This study was performed to identify the phase I metabolites of FYL‐67 using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The chemical structures were confirmed by comparison with corresponding chemical standards obtained internal. Primary elucidation of the metabolic pathway of FYL‐67 in vitro was performed using liver preparations (microsomes and hepatocytes) from rats and humans, and SD (Sprague Dawley, rat, rattus norvegicus) rats were used for the study of in vivo approach. To the end, two metabolites (M₁ and M₂) were detected after in vitro as well as in vivo experiments. Based on LC‐MS/MS analyses, the metabolites were demonstrated to be 5‐(aminomethyl)‐3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)oxazolidin‐2‐one (M₁) and 3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (M₂). Amide hydrolysis at acetyl group of FYL‐67 leading to the formation of M₁ was observed and suggested to play a major role in both in vivo and in vitro phase I metabolism of FYL‐67. M₁ was demonstrated to undergo a further oxidation to form M₂. In addition, the results indicated no species difference existing between rats and humans. The outcomes of our research can be utilized for the development and validation of the analytical method for the quantification of FYL‐67 as well as its metabolites in biological samples. Furthermore, it is helpful to conduct studies of pharmacodynamics and toxicodynamics. Copyright © 2015 John Wiley & Sons, Ltd.     

Crude extract of Rheum palmatum L. Induces cell cycle arrest S phase and apoptosis through mitochondrial‐dependent pathways in U‐2 OS human osteosarcoma cells

Cancer is the second cause of death in children. Osteosarcoma is the most common primary malignancy of solid bone cancer primarily affecting adolescents and young adults. In the Chinese population, the crude extract of Rheum palmatum L. (CERP) has been used for treating different diseases, including SARS, rheumatoid arthritis, coxsackievirus B3, and human colon cancer cell, pancreatic cancer. There are no reports on CERP and human osteosarcoma cells. The present study examined effects of CERP on cytotoxicity including cell cycle distribution and cell death (apoptosis) in U‐2 OS human osteosarcoma cells. CERP significantly induced S phase arrest in U‐2 OS cells in a dose‐dependent. CERP produced DNA damage and DNA condensation. Other effects of CERP were stimulation of ROS and Ca²⁺, mitochondria impairment, and activation of caspase‐3, ?8, and ?9. CERP increased the levels of Bax, Bak, Bad, cyclin B, Fas, PARP, GRP78, GADD153, AIF, Endo G, Calpain‐2, p21, and p27, but decreased the levels of Bcl‐2, BCL‐X, XIAP, Akt, CDC25A, CDK2, Cyclin A, and Cyclin E of U‐2 OS cells. It was also observed that CERP promoted the expression of AIF, Endo G, GADD153, and cytochrome c. These results indicate that CERP has anticancer effects in vitro and provide the foundation for in vivo studies of animal models of osteosarcoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 957–969, 2016.