Comparative Hazard Identification by a Single Dose Lung Exposure of Zinc Oxide and Silver Nanomaterials in Mice

Comparative hazard identification of nanomaterials (NMs) can aid in the prioritisation for further toxicity testing. Here, we assessed the acute lung, systemic and liver responses in C57BL/6N mice for three NMs to provide a hazard ranking. A silver (Ag), non-functionalised zinc oxide (ZnO) and a triethoxycaprylylsilane functionalised ZnO NM suspended in water with 2% mouse serum were examined 24 hours following a single intratracheal instillation (I.T.). An acute pulmonary inflammation was noted (marked by a polymorphonuclear neutrophil influx) with cell damage (LDH and total protein) in broncho-alveolar lavage fluid (BALF) after administration of both non-functionalised and functionalised ZnO. The latter also induced systemic inflammation measured as an increase in blood neutrophils and a decrease in blood lymphocytes. Exposure to Ag NM was not accompanied by pulmonary inflammation or cytotoxicity, or by systemic inflammation. A decrease in glutathione levels was demonstrated in the liver following exposure to high doses of all three nanomaterials irrespective of any noticeable inflammatory or cytotoxic effects in the lung. By applying benchmark dose (BMD) modeling statistics to compare potencies of the NMs, we rank functionalised ZnO ranked the highest based on the largest number of affected endpoints, as well as the strongest responses observed after 24 hours. The non-functionalised ZnO NM gave an almost similar response, whereas Ag NM did not cause an acute response at similar doses.     

Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO₂ NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO₂ NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO₂>TiO₂). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO₂ and SiO₂, while the embryonic stem cell test (EST) classified the TiO₂ NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO₂ NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO₂ NM-203 > SiO₂ NM-200 > TiO₂ NM-104 > TiO₂ NM-103). This ranking was different in the case of embryonic tissues, for which TiO₂ displayed higher toxicity compared with ZnO and SiO₂. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.     

Effects of manufactured nanomaterials on fishes: a target organ and body systems physiology approach

Manufactured nanomaterials (NM) are already used in consumer products and exposure modelling predicts releases of ng to low μg l(-1) levels of NMs into surface waters. The exposure of aquatic ecosystems, and therefore fishes, to manufactured NMs is inevitable. This review uses a physiological approach to describe the known effects of NMs on the body systems of fishes and to identify the internal target organs, as well as outline aspects of colloid chemistry relevant to fish biology. The acute toxicity data, suggest that the lethal concentration for many NMs is in the mg l(-1) range, and a number of sublethal effects have been reported at concentrations from c. 100 μg to 1 mg l(-1). Exposure to NMs in the water column can cause respiratory toxicity involving altered ventilation, mucus secretion and gill pathology. This may not lead, however, to overt haematological disturbances in the short term. The internal target organs include the liver, spleen and haematopoietic system, kidney, gut and brain; with toxic effects involving oxidative stress, ionoregulatory disturbances and organ pathologies. Some pathology appears to be novel for NMs, such as vascular injury in the brain of rainbow trout Oncorhynchus mykiss with carbon nanotubes. A lack of analytical methods, however, has prevented the reporting of NM concentrations in fish tissues, and the precise uptake mechanisms across the gill or gut are yet to be elucidated. The few dietary exposure studies conducted show no effects on growth or food intake at 10-100 mg kg(-1) inclusions of NMs in the diet of O. mykiss, but there are biochemical disturbances. Early life stages are sensitive to NMs with reports of lethal toxicity and developmental defects. There are many data gaps, however, including how water quality alters physiological responses, effects on immunity and chronic exposure data at environmentally relevant concentrations. Overall, the data so far suggest that the manufactured NMs are not as toxic as some traditional chemicals (e.g. some dissolved metals) and the innovative, responsible, development of nanotechnology should continue, with potential benefits for aquaculture, fisheries and fish health diagnostics.     

Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2)

The wide scale use of Zinc oxide (ZnO) nanoparticles in the world consumer market makes human beings more prone to the exposure to ZnO nanoparticles and its adverse effects. The liver, which is the primary organ of metabolism, might act as a major target organ for ZnO nanoparticles after they gain entry into the body through any of the possible routes. Therefore, the aim of the present study was to assess the apoptotic and genotoxic potential of ZnO nanoparticles in human liver cells (HepG2) and the underlying molecular mechanism of its cellular toxicity. The role of dissolution in the toxicity of ZnO nanoparticles was also investigated. Our results demonstrate that HepG2 cells exposed to 14-20 μg/ml ZnO nanoparticles for 12 h showed a decrease in cell viability and the mode of cell death induced by ZnO nanoparticles was apoptosis. They also induced DNA damage which was mediated by oxidative stress as evidenced by an increase in Fpg sensitive sites. Reactive oxygen species triggered a decrease in mitochondria membrane potential and an increase in the ratio of Bax/Bcl2 leading to mitochondria mediated pathway involved in apoptosis. In addition, ZnO nanoparticles activated JNK, p38 and induced p53(Ser15) phosphorylation. However, apoptosis was found to be independent of JNK and p38 pathways. This study investigating the effects of ZnO nanoparticles in human liver cells has provided valuable insights into the mechanism of toxicity induced by ZnO nanoparticles.     

可见光响应的CdTe/TiO2复合纳米颗粒体外PDT灭活HL60细胞的研究

文章采用溶胶凝胶法制备核壳CdTe/TiO_2复合纳米颗粒,探讨了该复合纳米颗粒体外PDT对HL60细胞的灭活作用。通过扫描电镜(TEM)、X射线光电子衍射仪(XPS)对CdTe/TiO_2进行表征。文中,用紫外可见光吸收光谱(UV-vis)测得尺寸为2-5 nm的CdTe QDs吸收峰为460 nm。研究表明,CdTe/TiO_2复合纳米颗粒尺寸在80 nm左右,其吸收光谱相较于TiO_2的光响应区拓展至可见光区。将CdTe/TiO_2与HL60细胞进行共同孵育,采用CCK-8法研究了其在暗室条件下细胞的生长情况和浓度对细胞相对存活率的影响以及在不同浓度的CdTe/TiO_2复合纳米颗粒PDT后的细胞活性。实验结果表明:在共同孵育16 h后CdTe/TiO_2对HL60细胞的毒性最强,10~320μg/mL浓度的CdTe/TiO_2样品对HL60细胞均具有较强的灭活作用。当添加CdTe/TiO_2样品浓度为320μg/mL时,光照1 h后PDT灭活效率达到87.7%。     

Leishmania donovani: Oral efficacy and toxicity of formycin B in the infected hamster

Formycin B, a structural analog of inosine, was evaluated as an orally administrable antileishmanial agent. Against Leishmania donovani in hamsters, it achieved an 85–92% reduction in numbers of parasites in livers of infected animals after oral administration at 13 mg/kg/day for 4 days. Its efficacy by oral administration was approximately four to eight times that by intramuscular administration and four times that of the positive control drug Glucantime by intramuscular administration. The levels of formycin B in serum after the final oral administration of 26 mg/kg/day were 1.4 μg/ml at 1 hr and 0.3 μg/ml at 2 hr. The concentration in liver was greater (9.0 μg/ml at 1 hr) and declined more slowly. With this latter dosage or with 104 mg/kg/day there was no acute toxicity of formycin B to bone marrow or formed elements of the blood. The only statistically significant toxicity to the liver was a doubling of serum total bilirubin levels. Comparison of the in vivo efficacy of formycin B against L. donovani to the mild acute toxicity of the drug suggests that formycin B has potential as an oral agent against visceral leishmaniasis.     

Genotoxicity of the coating lacquer on food cans, bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE

The epoxy resin bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE (BADGE·2HCl), were examined for their genotoxicity in the micronucleus test (MNT) with human peripheral blood lymphocytes in vitro, in presence and in absence of an exogenous metabolizing system S9 rat liver. The treatment was done using different compound concentrations up to cytotoxic doses. The concentrations tested ranged between 12.5 to 62.5 μg/ml of BADGE, 12.5 to 62.5 μg/ml of first BADGE hydrolysis product (BADGE·H₂O), 25.0 to 100.0 μg/ml of second BADGE hydrolysis product (BADGE·2H₂O) and 6.25 to 50.0 μg/ml of BADGE·2HCl. These compounds are able to induce both cytotoxic and genotoxic effects, as revealed by the increases observed in cytokinesis block proliferation index (CBPI) and in micronuclei (MN) frequencies, respectively.     

Genotoxicity of the coating lacquer on food cans, bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE

The epoxy resin bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE (BADGE.2HCl), were examined for their genotoxicity in the micronucleus test (MNT) with human peripheral blood lymphocytes in vitro, in presence and in absence of an exogenous metabolizing system S9 rat liver. The treatment was done using different compound concentrations up to cytotoxic doses. The concentrations tested ranged between 12.5 to 62.5microg/ml of BADGE, 12.5 to 62.5microg/ml of first BADGE hydrolysis product (BADGE.H(2)O), 25.0 to 100.0microg/ml of second BADGE hydrolysis product (BADGE.2H(2)O) and 6.25 to 50.0microg/ml of BADGE.2HCl. These compounds are able to induce both cytotoxic and genotoxic effects, as revealed by the increases observed in cytokinesis block proliferation index (CBPI) and in micronuclei (MN) frequencies, respectively.     

Nanomaterials in the Environment Acquire an “Eco‐Corona” Impacting their Toxicity to Daphnia Magna—a Call for Updating Toxicity Testing Policies

Nanomaterials (NMs) are particles with at least one dimension between 1 and 100 nm and a large surface area to volume ratio, providing them with exceptional qualities that are exploited in a variety of industrial fields. Deposition of NMs into environmental waters during or after use leads to the adsorption of an ecological (eco‐) corona, whereby a layer of natural biomolecules coats the NM changing its stability, identity and ultimately toxicity. The eco‐corona is not currently incorporated into ecotoxicity tests, although it has been shown to alter the interactions of NMs with organisms such as Daphnia magna (D. magna). Here, the literature on environmental biomolecule interactions with NMs is synthesized and a framework for understanding the eco‐corona composition and its role in modulating NMs ecotoxicity is presented, utilizing D. magna as a model. The importance of including biomolecules as part of the current international efforts to update the standard testing protocols for NMs, is highlighted. Facilitating the formation of an eco‐corona prior to NMs ecotoxicity testing will ensure that signaling pathways perturbed by the NMs are real rather than being associated with the damage arising from reactive NM surfaces “acquiring” a corona by pulling biomolecules from the organism's surface.     

In vitro and in vivo effects of the combination of myricetin and miconazole nitrate incorporated to thermosensitive hydrogels,on C. albicans biofilms

BackgroundCandida albicans-related infections are common infections in clinic, among which biofilm-associated infections are most devastating and challenging to overcome. Myricetin (MY) is a plant-derived natural product with various pharmacological activities. Its anti-biofilm effect against C. albicans and its ability to increase the antifungal effect of miconazole nitrate (MN) were unclear and yet need to be explored.Hypothesis/PurposeIn this study the anti-biofilm effect of MY and its ability to increase the antifungal effect of MN were investigated in vitro and in vivo.Study design and methodsMY or/and MN were incorporated into a thermosensitive hydrogel (TSH) of poloxamer. The safety of MY or/and MN loaded TSHs towards human umbilical vein endothelial cells (HUVEC) was evaluated by a MTT assay and the in vivo safety towards mice knees was confirmed by histopathological examination. The anti-biofilm effect of MY and its ability to increase the antifungal effect of MN were investigated in vitro with C. albicans ATCC 10231 by broth microdilution method, crystal violet staining and scanning electron microscopy (SEM), as well as in vivo in an established mouse model of periprosthetic joint infection (PJI) by SEM, histological analysis, microorganism culture and detection of the serum levels of interleukin-6 (IL-6). The mechanism of action of MY was analyzed by qRT-PCR assay with C. albicans SC5314.ResultsOur results showed that MY and MN incorporated into TSHs exhibited good stability and safety, excellent temperature sensitivity and controlled drug release property. MY (5-640 µg/ml) exhibited no effect on C. albicans cell viability and MY (≥80 µg/ml) showed a significantly inhibitory effect on biofilm formation. MIC₅₀ (the lowest concentrations of drugs resulting in 50% decrease of C. albicans growth) and MIC₈₀ (the lowest concentrations of drugs resulting in 80% decrease of C. albicans growth) of MN were respectively decreased from 2 µg/ml to 0.5 µg/ml and from 4 µg/ml to 2 µg/ml when used in combination with MY (80 µg/ml). The mouse PJI was effectively prevented by MY and MN incorporated into TSH.ConclusionsLocal application of MY and MN incorporated into TSH might be useful for clinical biofilm-associated infections.     

Effects of Different Calcium, Magnesium, and Zinc Concentrations Supplemented on Hepatopancreatic Cell Proliferation of Kuruma Prawn, Penaeus japonicus

The effects of different calcium (Ca(2+)), magnesium (Mg(2+)), and zinc (Zn(2+)) concentrations supplemented on hepatopancreatic cell proliferation of kuruma prawn, Penaeus japonicus was studied. The culture system consists of medium 199 (M 199) supplemented with 0.060 mol/L NaCl, 1.011 g/L glucose, 1,000 UI/ml penicillin, 1,000 μg/ml treptomycin, heat inactivated fetal calf serum (FCS) 20% for primary cell culture and 10% for subculture. The RNA/DNA ratio of the cell cultures was measured. The results show that the cell division of hepatopancreatic cells of P. japonicus was enhanced by the optimal concentration of inorganic salt (Ca(2+), 1.0 g/L; Mg(2+), 5.0 g/L; Zn(2+), 80 μg/L). The hepatopancreatic cell culture system and improved culture conditions described here will be very useful for in vitro experiments to study viruses responsible for infections in shrimp leading to tremendous economic losses.     

Neuromelanins of Human Brain Have Soluble and Insoluble Components with Dolichols Attached to the Melanic Structure

Neuromelanins (NMs) are neuronal pigments of melanic-lipidic type which accumulate during aging. They are involved in protective and degenerative mechanisms depending on the cellular context, however their structures are still poorly understood. NMs from nine human brain areas were analyzed in detail. Elemental analysis led to identification of three types of NM, while infrared spectroscopy showed that NMs from neurons of substantia nigra and locus coeruleus, which selectively degenerate in Parkinson’s disease, have similar structure but different from NMs from brain regions not targeted by the disease. Synthetic melanins containing Fe and bovine serum albumin were prepared to model the natural product and help clarifying the structure of NMs. Extensive nuclear magnetic resonance spectroscopy studies showed the presence of dolichols both in the soluble and insoluble parts of NM. Diffusion measurements demonstrated that the dimethyl sulfoxide soluble components consist of oligomeric precursors with MWs in the range 1.4–52 kDa, while the insoluble part contains polymers of larger size but with a similar composition. These data suggest that the selective vulnerability of neurons of substantia nigra and locus coeruleus in Parkinson’s disease might depend on the structure of the pigment. Moreover, they allow to propose a pathway for NM biosynthesis in human brain.     

Genotoxic evaluation of the antimalarial drug,fansidar, in cultured human lymphocytes

Fansidar (pyrimethamine-sulfadoxine) has been used extensively worldwide for the treatment of chloroquine resistant Plasmodium falciparum malaria, toxoplasmosis and Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. Because of the wide usage of pyrimethamine-sulfadoxine in developing countries and the lake of information from open literature and reports from manufacturers about the genotoxicity of such antimalarial drug, the present work was suggested. The possible genetic toxicity of fansidar has been evaluated in human peripheral blood lymphocyte cultures. The frequencies of sister-chromatid exchanges (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests covering a wide range of induced genetic damage as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up by using blood samples from two healthy donors and the treatment was done using different fansidar concentrations ranging from 1:20 to 10:200 g/ml. From our results, it appears that this drug is able to induce moderate genotoxic effects, as revealed by the increases found in SCE and MN frequencies in cultures from the two donors at the two highest concentrations tested (5:100 and 10:200 g/ml). In addition, cyotoxic/cytostatic effects of fansidar were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI). Our findings suggest that the use of this drug should be restricted to situations where other antimalarial drugs cannot be used. The drug should never be given to pregnant women.     

Biomethanation of spent wash: Heavy metal inhibition of methanogenesis in synthetic medium

Five heavy metals detected in distillery waste were lead (1.0–8.8 μg/ml), copper (1.7–15.7 μg/ml), zinc (3.1–11.8 μg/ml), iron (36.0–43.5 μg/ml), and manganese (3.0–5.1 μg/ml). Their toxicity to biomethanogenesis in a synthetic medium containing 1% sodium acetate, propionate, or butyrate was measured by batch fermentation, after cultivating the bacterial biomass semicontinuously. Lead, copper, and zinc in decreasing order were found to be toxic to biomethanogenesis. Lead at the concentration of 10 μg/ml completely stopped methane production. Iron did not produce any notable change in the process while manganese stimulated the rate of methane production. The toxicity of lead, copper, and zinc to methanogenic bacteria and methane production was dose-dependent but the growth of acetogenic bacteria was impaired at higher concentrations (2.5–10.0 μg/ml) of lead, copper, and zinc. Manganese stimulated the growth of only methanogenic bacteria, but not that of non-methanogenic bacteria or acetic acid production. The reduction in the synthesis of acetic acid via butyrate was more in the presence of these three metals than the synthesis of this acid via propionate.     

Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test. I. Theoretical aspects

A decrease in the cytokinesis-block proliferation index (CBPI) or replication index (RI) is routinely used to determine cytotoxicity of a test compound and therefore the choice of its appropriate test concentration for the in vitro micronucleus (MN) test conducted in the presence of cytochalasin B. As a number of laboratories prefer to conduct the in vitro MN test in the absence of cytochalasin B, it is important that selected test concentrations, based on cytotoxicity, should be similar to what they would have been if cytochalasin B had been used, and should be relevant of a true cytotoxicity. By using models to analyse the dynamics of the cell cultures with and without cytochalasin B we have compared different methods for evaluation of cytotoxicity, and demonstrate that relative decrease in population doubling or relative increase in cell counts are the most appropriate measures of cytotoxicity to compare with reduction in CBPI or RI.     

人主动脉硫酸乙酰肝素蛋白聚糖对培养人的脐静脉内皮细胞生长的影响

为探讨硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对内皮细胞生长的作用,用解聚提取及离子交换柱层析法分离出人主动脉ＨＳＰＧ,用倒置显微镜、细胞计数、及￣３Ｎ－ＴｄＲ参入观察其对培养的第一代人脐静脉内皮细胞（ｈＵＶＦＣ）生长的影响。结果发现：（１）倒置显微镜下观察,加入ＨＳＰＧ（１．７０μｇ已糖醛酸／ｍｌ）的ｈＵＶＥＣ生长密度高于对照组（未加ＨＳＰＧ）．（２）随着培养时间增加（２４,４８及７２ｈ）．根据细胞计数计算出同一剂量的ＨＳＰＧ（１７．０μｇ已糖醛酸／ｍｌ）对ｈＵＶＥＣ的促增殖％增高（分别为１４％,３０％及３７％）。（３）随着加入ＨＳＰＧ浓度的升高（４．３,８．５及１７．０μｇ已糖醛酸／ｍｌ．培养７２ｈ）．根据￣３Ｈ－ＴｄＲ参入计算出ＨＳＰＧ对ｈＵＶＥＣ的促增殖％亦增高（分别为４９％,７１％及９８％）。故人主动脉ＨＳＰＧ对培养的人脐静脉内皮细胞有促增殖作用。     

金黄色葡萄球菌肠毒素A对H22小鼠移植瘤的抑制作用

旨在探讨金黄色葡萄球菌肠毒素A(SEA)在KM鼠体内的抑瘤效果.建立H22荷瘤小鼠模型,将20只小鼠随机分为对照组(生理盐水)、低剂量组(15 μg/ml)、中剂量组(25 μg/ml)和高剂量组(50 μg/ml),观察肿瘤生长趋势,计算抑瘤率,通过ELISA检测IL-2的含量.结果显示,给药组肿瘤生长趋势均较对照组缓慢;对照组、低剂量组、中剂量组、高剂量组的抑瘤率分别为0,28.9%,34.0%,51.0%(P<0.05);血清中IL-2含量分别为58.9 pg/ml、83.6 pg/ml、91.8 pg/ml、118.1 pg/ml.金黄色葡萄球菌肠毒素A(SEA)可抑制H22肿瘤的生长,并上调IL-2的分泌.     

High-performance liquid chromatographic determination of (−)-β-d-2,6-diaminopurine dioxolane and its metabolite,dioxolane guanosine,using ultraviolet and on-line radiochemical detection

(−)-β-d-2,6-Diaminopurine dioxolane (DAPD) and its metabolite dioxolane guanosine (DXG) have potent activity against hepatitis B virus and HIV, in vitro. A reversed-phase HPLC analytical method using UV and on-line radiochemical detection for the determination of DAPD and DXG in monkey serum and urine is described in this report. Retention times for DXG, DAPD and internal standard (2′,3′-didehydro-2′ deoxythymidine, D4T) were 5.0, 6.0 and 13.0 min, respectively. The extraction recovery was greater than 97% for DAPD and 94% for DXG. The limit of quantitation for UV detection was 100 ng/ml and 125 ng/ml for DXG and DAPD in monkey serum. The standard curves were linear from 0.1 μg/ml to 5 μg/ml for DXG and 0.125 μg/ml to 5 μg/ml for DAPD. For radiochemical detection, calibration curves of standard solutions of DAPD and DXG were linear in the range of 3500 Bq to 32 000 Bq and 7500 Bq to 60 000 Bq. The intra- and inter-day relative standard deviations were less than 7.2% using UV and less than 8.6% using on-line radiochemical detection. The HPLC method was applied to serum and urine samples collected from a male rhesus monkey that was administered 33.3 mg/kg DAPD with 200 μgCi of [³H]DAPD intravenously.