Preparation of dry reconstituted liposomal powder by freeze-drying at room temperature

The aim of this study was to develop a novel, one-step method of liposome preparation by freeze-drying at room temperature as well as to investigate the physicochemical properties of dry reconstituted liposomal powder that was prepared. The method was based on utilizing sublimation of a volatile solid inert carrier, that is, chlorobutanol hemihydrate (CBN), instead of ice, which was less sophisticated and simpler than the conventional freeze-drying process. The optimum conditions used in the sublimation process of CBN were a temperature of 25–30°C and a pressure of 1.5–2.0 mBar for 8 hours. The influence of various parameters, such as type, particle size, and ratio of sugar lyoprotectant (i.e., mannitol or sucrose) and CBN to lipid on reconstitution time, liposome size, zeta potential, vesicle type, and lamella structure of reconstituted liposomes, were studied. The results revealed that the obtained liposomes were oligolamellar vesicles with particle sizes ranging from 400 to 1,000?nm. Type and ratio of sugar and CBN to lipid were found to significantly affect the reconstitution time. On the other hand, liposome size was independent of type of sugar and ratio of CBN to lipid, yet became smaller at higher sugar-to-lipid ratio and smaller sugar and CBN size. In all cases, traces of residual solvents were definitely below the acceptable limit.     

Cannabinol modulates neuroprotection and intraocular pressure: A potential multi-target therapeutic intervention for glaucoma

ObjectivesGlaucoma is characterized by progressive damage of the retinal ganglion cells (RGCs), resulting in irreversible vision loss. Cannabinoids (CBs) ameliorate several factors that contribute to the progression of glaucoma, including increased intraocular pressure (IOP), degeneration of RGC and optical nerve (ON) damage. However, a direct correlation of specific CBs with the molecular events pertaining to glaucoma pathology is not well established. Therefore, this study aims to evaluate the role of cannabinol (CBN) on RGC protection, modulation of IOP, and its effects on the level of extracellular matrix (ECM) proteins using both in vitro and in vivo models of glaucoma.Methods and resultsWhen exposed to elevated hydrostatic pressure, CBN, in a dose-dependent manner, protected differentiated mouse 661W retinal ganglion precursor-like cells from pressure-induced toxicity. In human trabecular meshwork cells (hTM), CBN attenuated changes in the ECM proteins, including fibronectin and α-smooth muscle actin (α-SMA), as well as mitogen-activated protein kinases (phospho-ERK1/2) in the presence or absence of transforming growth factor-beta 2 (TGF-β₂) induced stress. Ocular pharmacokinetic parameters were evaluated post-intravitreal (IVT) CBN delivery in vivo. Furthermore, we demonstrated that IVT-administered CBN improved pattern electroretinogram (pERG) amplitudes and reduced IOP in a rat episcleral vein laser photocoagulation model of glaucoma.ConclusionCBN promotes neuroprotection, abrogates changes in ECM protein, and normalizes the IOP levels in the eye. Therefore, our observations in the present study indicate a therapeutic potential for CBN in the treatment of glaucoma.     

Study on the interaction between cannabinol and DNA using acridine orange as a fluorescence probe

The interaction between cannabinol (CBN) and herring‐sperm deoxyribonucleic acid was investigated by using acridine orange as a fluorescence probe in this work. UV‐Vis spectroscopy, fluorescence spectroscopy, and DNA melting techniques were used. The fluorescence of DNA acridine orange was quenched by CBN. The results indicated that CBN can bind to DNA. The binding constant for the CBN and herring‐sperm deoxyribonucleic acid was obtained at 3 temperatures, respectively. Results of molecular docking corroborated the experimental results obtained from spectroscopic investigations. The influence of ionic strength on the fluorescence properties was also investigated. The thermodynamic results indicated that hydrophobic interaction played a major role in the binding between CBN and DNA.     

基于多种微生物的毒性检测MTOXPlate冻干板制备及优化

[目的]制备基于多种微生物的生物毒性检测MTOXPlate冻干板,从而简化操作程序,使其能更灵敏、方便、快速地进行生物毒性测试.[方法]采用真空冷冻干燥法将指示微生物固定在微孔板中.以存活率为指标,利用荧光分光光度法确定冻干保护剂的组成.通过分析毒性响应灵敏度对检测条件进行优化.[结果]MTOXPlate的冻干保护剂组成为8％海藻糖、2％葡萄糖、2％甘露醇、1％谷氨酸钠.在此组成下,11株菌株的平均存活率最高,可达89.41％.复活介质与测试样品同时加入,在缩短了测试时间的同时也提高了检测灵敏度.该冻干板具有良好的灵敏度和重现性,能较好地满足污染物生物毒性测试的需要.[结论]MTOXPlate冻干板可作为一种新型的生物毒性测试技术,在生态毒理检测领域推广应用.     

Causal Assessment of Biological Impairment in the Little Floyd River,Iowa, USA

An assessment of biological impairment in the Little Floyd River (Iowa, USA) was based on evidence of three characteristics of causation: co-occurrence, preceding causation, and sufficiency. Evidence of the physical interaction of the probable causes and the biota, resulting alterations to the biota, as well as the time order of the cause and the effect were consistent within the assessment, but the evidence for these causal characteristics did not discriminate among probable causes or other causes. Deposited sediment, low dissolved oxygen, heat stress, and ammonia toxicity are the probable causes of impaired biological condition in the Little Floyd River compared with other rivers in the ecoregion. Less likely causes are suspended sediment, altered basal food resources, and flow alteration. Very unlikely causes are pH shifts, total dissolved solids, Cyprinus carpio (an invasive species), metal toxicity, and pesticides. Data were insufficient to assess salinity or other toxicants. The assessment was used to develop a recovery plan for the stream. This assessment demonstrates that, even when there are many candidate causes and uncertainties are substantial, the probable causes of biological impairments can be determined with enough certainty to inform decision-making to address environmental problems.     

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at −5 °C for 30 min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60 min at 25 °C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16 Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25 °C for 30 min, the maturation rate decreased in solution with 0.51 Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40 Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25 °C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at −5 °C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.     

Antitumor activity and toxicity of peroxo heteroligand vanadates(V) in relation to biochemistry of vanadium

A selected set of 14 V(V) complexes was tested for toxicity and antitumor activity against L1210 murine leukemia, to examine the biological properties of peroxoheteroligand vanadates(V) of the formula (NH4)4[O(VO(O2)2)2], M3I[VO(O2)2C2O4], and MI[VO(O2)L], L = malate, citrate, iminodiacetate, nitrilotriacetate, and EDTA. The x-ray structure is known for five of these compounds. A relationship has been found between the chemical composition and the biological activity (antitumor activity-toxicity) of these complexes. Activity in the L1210 system is defined as greater than or equal to 25% increase in life span, and this was seen with (NH4)4[O(VO(O2)2)2]; M3[VO(O2)2(C2O4)]2H2O, M = K, NH4; and NH4[VO(O2)Malato]H2O. These observations are important for the biochemistry of vanadium. The special nature of electron transfer within the V(V)-peroxo moiety is proposed to be responsible for this phenomenon. Peroxo heteroligand vanadates(V) therefore represent a model system for studying some biochemical interactions of vanadium in living matter.     

Environmental Assessment of Two Waste Incineration Strategies for Central Norway (10 pp)

Background, Aims and Scope A strategy of local incineration with 17 small incinerators (Scenario L) is compared to a strategy of 3 centralized waste incinerators (Scenario C) for the region of Central Norway, in order to assess differences in environmental performance. Rough calculations of costs are also included. The functional unit is the treatment of municipal, commercial and special waste not recycled, plus the heating of a specific number of households, for the period of 2002 to 2020. Methods Data on large and small scale incinerators were obtained from technology providers. LCA databases were used for transportation and heating, while cost estimates and Norwegian input-output tables were used for the construction of the facilities. The CML2 method was used to evaluate the impacts. Results and Discussion Transportation is a major contributor to aquatic toxicity and acidification as well as CO2 emissions. Impacts from construction are considerable for photochemical oxidation, while incineration is important for terrestrial toxicity and CO2 emissions. Conclusion Construction and operation of treatment facilities are, together with transportation, the main processes making a difference between the two strategies. Substantial gains will come from the reduction in transportation need when introducing a local incineration strategy. When considering a time span of two decades, the centralized scenario is about 2.5 to 5 times the impact potential of the local scenario for most impact categories, in terms of cumulative results. Cost estimates for the two solutions support these findings, as cumulated costs also favors a local solution. Recommendation and Outlook Transportation is a major contributor to several impact categories, and especially the transportation of special waste should become more efficient in terms of transportation distances. Cost estimates support the environmental assessment, but a more comprehensive economic study of the system would be valuable.     

Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes

In this study, tetrahydrocannabinols (THCs) were mainly oxidized at the 11-position and allylic sites at the 7alpha-position for Delta(8)-THC and the 8beta-position for Delta(9)-THC by human hepatic microsomes. Cannabinol (CBN) was also mainly metabolized to 11-hydroxy-CBN and 8-hydroxy-CBN by the microsomes. The 11-hydroxylation of three cannabinoids by the microsomes was markedly inhibited by sulfaphenazole, a selective inhibitor of CYP2C enzymes, while the hydroxylations at the 7alpha-(Delta(8)-THC), 8beta-(Delta(9)-THC) and 8-positions (CBN) of the corresponding cannabinoids were highly inhibited by ketoconazole, a selective inhibitor of CYP3A enzymes. Human CYP2C9-Arg expressed in the microsomes of human B lymphoblastoid cells efficiently catalyzed the 11-hydroxylation of Delta(8)-THC (7.60 nmol/min/nmol CYP), Delta(9)-THC (19.2 nmol/min/nmol CYP) and CBN (6.62 nmol/min/nmol CYP). Human CYP3A4 expressed in the cells catalyzed the 7alpha-(5.34 nmol/min/nmol CYP) and 7beta-hydroxylation (1.39 nmol/min/nmol CYP) of Delta(8)-THC, the 8beta-hydroxylation (6.10 nmol/min/nmol CYP) and 9alpha,10alpha-epoxidation (1.71 nmol/min/nmol CYP) of Delta(9)-THC, and the 8-hydroxylation of CBN (1.45 nmol/min/nmol CYP). These results indicate that CYP2C9 and CYP3A4 are major enzymes involved in the 11-hydroxylation and the 8-(or the 7-) hydroxylation, respectively, of the cannabinoids by human hepatic microsomes. In addition, CYP3A4 is a major enzyme responsible for the 7alpha- and 7beta-hydroxylation of Delta(8)-THC, and the 9alpha,10alpha-epoxidation of Delta(9)-THC.     

Effect of cannabinoids on spermatogenesis in vivo: a cytological study

The cytogenetic effects of delta 9-tetrahydrocannabinol (THC) and cannabinol (CBN) (10 mg/kg) were investigated in hybrid mice of genotype (C57BL x C3H)F1. Mice were treated for 5 consecutive days with the specific cannabinoid; 16 days after the last treatment the meiotic cells were evaluated. Analysis of the spermatocyte bivalents at the first meiotic metaphase failed to reveal any numerical or structural abnormality. Contrary to previous reports we failed to find any major meiotic abnormalities associated with THC and CBN treatments. There was no evidence of ring or chain figures. The centromeric banding procedure (C banding) was used to support observations of Giemsa stained cells. It has previously been reported that cannabinoids suppress the incorporation of 3H-uridine into pachytene spermatocytes and round spermatids; the absence of chromosome aberrations in the present study suggests that an adverse effect appears to be present, but may be at a level other than cytogenetic.     

A study of biological fluids by ultrasound coagulography

A new methodology: the method and technical means for studying the coagulation of blood and the acoustic properties of other biological fluids (ultrasound coagulography) was developed. The complex of devices is based on the principle of measuring the rate of ultrasound in a medium studied by means of the impulse method. Arguments for the applicability of the method for studying the process of blood coagulation are adduced, special acoustic chambers were designed, and algorithms and computer programs for immediate processing of the data were developed. The field of application is biology and medicine: studies of the acoustic properties of biological fluids (e.g., blood coagulation), rheological properties, and viscosity of the object for the diagnosis of some diseases. The results of clinical assays of blood coagulation in healthy persons are presented.     

Microbioluminescent study of the general toxicity and mutagenicity of pollutants

Bacterial bioluminescence was applied to detection of general toxicity (MIT test) and genotoxicity (SOS-lux test) of some chemicals, seawater, and fresh water. The SOS-induced luminescence of E. coli WP2s (cda::luxCDABE) cells was higher than in E. coli C 600 (cda::luxCDABE) at 37 degrees C and pH 6.5. The mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide determined from the induction of E. coli WP2s cell luminescence was detected at lower concentrations than in the assessment of reversion frequencies. General toxicity was demonstrated by using luminescence inhibition for hydrogen peroxide, Zn2+, and Cd2+ at low concentrations. Regions of the Krasnodar Krai where sea and fresh waters exerted toxic action on luminescence were determined by the microbioluminescent method.     

无瓣海桑果叶及其浸提液的生物急性毒性研究

为了探讨无瓣海桑的生物毒性作用,采用哺乳类动物大鼠,国际规定的标准毒性实验用鱼斑马鱼以及红树林内主要鱼种弹涂鱼为受体,进行了大鼠急性经口毒性固定剂量毒性反应观察,斑马鱼和弹涂鱼的改进寇氏法毒性实验以及半致死量浓度(LC₅₀)和95%可信限计算,结果表明:在5000 mg/kg体重下,无瓣海桑的果实和叶子浸提液都未对大鼠产生任何毒性表现,14 d的死亡率为0,半致死量(LD₅₀)大于5000 mg/kg体重,属于无毒物质;对鱼类毒性,相同鱼种下,果实水浸提液毒性强于叶子水浸提液,相同水浸提液下,弹涂鱼对毒性敏感度超过斑马鱼,半致死浓度24 hLC₅₀>4 8 hLC₅₀>72 hLC₅₀>96 hLC₅₀>1000 mg·L^-1,属于低毒物质.根据以上初步得出无瓣海桑不会对周围生物造成毒性危害.     

Simple and sensitive determination of Delta(9)-tetrahydrocannabinol, cannabidiol and cannabinol in hair by combined silylation, headspace solid phase microextraction and gas chromatography-mass spectrometry

A new method for determination of Delta(9)-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair based on alkaline hair hydrolysis, extraction by iso-octane, combined derivatization with N,O-bis-(trimethylsilyl)-trifluoroacetamide and headspace solid phase microextraction of the extract residue, and gas chromatography-mass spectrometry was developed and evaluated. The limits of detection of the three compounds were 0.01-0.02 ng/mg. The method was routinely applied to more than 250 hair samples. In 77 positive samples, the concentrations ranged from LOD to 4.2 ng/mg for THC (mean 0.49 ng/mg), to 12.1 ng/mg for CBD (mean 0.37 ng/mg) and to 0.85 ng/mg for CBN (mean 0.12 ng/mg) using a sample amount of 30 mg. The frequently observed increase of the segmental drug concentrations from proximal to distal is explained by progressive accumulation in the hair shaft from sebum or side stream smoke.     

Microcalorimetric investigation of the toxic action of Cd(2+) on Rhizopus nigricans growth

The microcalorimetric bioassay for acute cellular toxicity is based on metabolic heat production from cultured cells. Microcalorimetry is a quantitative, inexpensive, and versatile method for toxicology research. The biological response to toxicants is the inhibition of the heat production rate in cells and toxicity is expressed as the concentration of toxicant that is 50% effective in this inhibition (IC(50)). In this paper, the effect of Cd(2+) on Rhizopus nigricans growth was investigated at 25 degrees C. The relationship between growth rate constants (k) and concentration of Cd(2+) (C) shows a logarithmic normal distribution, and described as k=1. 2742x10(61)exp[-1.810x10(-3)(C+283.0)(2)], and IC(50) is 0.72 microg/ml. These signals are readily obtained by an LKB 2277-204 heat conduction microcalorimeter.     

Plan for an Integrated Human and Environmental Risk Assessment in the S. Domingos Mine Area (Portugal)

The need for the integration of the assessment of human and ecological risks in contaminated areas, such as derelict mines, widely increases. The risk assessment process is becoming a powerful tool to provide sound scientific bases for decision-making processes. In Portugal, the risk assessment process is in its early years and the lack of multidisciplinary teams of experts is frequently mentioned as the main obstacle to its implementation. Therefore, the majority of the reclamation actions are based on impact assessment studies that usually are characterized by few biological and toxicological considerations. In order to account for some of these constraints, the ecological risk assessment framework proposed by the U.S. Environmental Protection Agency was used to plan the assessment of human and ecological risks posed by the high concentrations of metals scattered in the vicinity of S. Domingos mine, a cuprous pyrite mine located in the Southeast Alentejo (Portugal). This study presents the problem formulation phase of the assessment. It includes all the scientific information available for the area, a conceptual model, and an analysis plan for the risk assessment process. Following a tiered approach, several tasks were planned in order to acquire chemical, toxicological, and ecological information, in order to compensate for the lack of toxicity data for site-specific species.     

计算系统生物学:理论、方法及在药物研发中的应用

计算系统生物学是一个多学科交叉的新兴领域,旨在通过整合海量数据建立其生物系统相互作用的复杂网络。数据的整合和模型的建立需要发展合适的数学方法和软件工具,这也是计算系统生物学的主要任务。生物系统模型有助于从整体上理解生物体的内在功能和特性。同时,生物网络模型在药物研发中的应用也越来越受到制药企业以及新药研发机构的重视,如用于特异性药物作用靶点的预测和药物毒性评估等。该文简要介绍计算系统生物学的常见网络和计算模型,以及建立模型所用的研究方法,并阐述其在建模和分析中的作用及面临的问题和挑战。     

Nematode-based biomarkers as critical risk indicators on assessing the impact of silver nanoparticles on soil ecosystems

Soil contamination caused by silver nanoparticles (AgNPs) released from sewage treatment plants (STPs) is of great public concern. Understanding the relationships between the physicochemical properties of AgNPs and their toxicity is critical for environmental and health risk analysis. Here we presented an approach for rapidly screening and assessing the potential toxicity risk of AgNPs in general and sludge-treated soils based on the nematode Caenhorhabditis elegans-based probabilistic risk assessment framework. The soil environmental risks were estimated depending on the characteristics of AgNPs and geographic regions. We assessed the risk for soils exceeding a threshold of C. elegans neurotoxicity based on the statistical models. Our results indicated that locomotion inhibition of C. elegans was depending on surface properties, diameter, and exposure time of AgNPs. Here we showed that the overall sewage sludge-released AgNPs-associated soil contamination risk was very low among Europe, U.S., and Switzerland. However, large production and widespread use of AgNPs are highly likely to pose long-term ecotoxicity risk on general and sludge-treated soils, particularly for 26 nm citrate-coated AgNPs. Our approach of integrating probabilistic risk model and C. elegans-based ecological indicator provides an effective tool to rapidly screen and assess the impacts of STPs-released AgNPs on soil environment. We suggest that C. elegans as a proxy for estimating soil risk metrics can help develop methods of management for mitigating the metal NPs-induced toxicity on terrestrial ecosystems.     

Toxicity of Fluoranthene and Its Biodegradation by Cyclotella caspia Alga

Fluoranthene Is one of the polynuclear aromatic hydrocarbons with four benzene rings. Because of Its toxicity, mutagenlclty, and carclnogenlclty, fluoranthene Is on the black lists of 129 and 68 priority pollutants established by US Environmental Protection Agency and the People＇s Republic of China, respectively. In recent years, the amount of fluoranthene In the aquatic environment has been Increasing with Increases In anthropogenlc discharge. Based on the biological investigation of tidal water In the Futlan mangrove, Cycioteila ~aspla was selected as the dominant algal species to determine the toxicity of fluoranthene towards C. caspla alga and to Investigate the blodegradatlon of fluoranthene by C. caspla under pure culture. The toxicity experiment showed that the 96-h EC50 value for fluoranthene was 0.2 mg/mL. Four parameters, namely C. caspla algal growth rate, chlorophyll （Chl） a content, cell morphology, and superoxlde dlsmutase （SOD） activity, were chosen as Indices of toxicity and were measured at 6 d （144 h）. The results showed that： （Ⅰ） the toxicity of fluoranthene towards C. caspla alga was obvious; （Ⅱ） C. caspla algal growth rate and Chl a content decreased with Increasing concentrations of fluoranthene; and （Ⅲ） the rate of cell deformation and SOD activity Increased with Increasing concentrations of fluoranthene. The blodegradatlon experiment showed that： （Ⅰ） the rate of physical degradation of fluoranthene was only 5.86%; （Ⅱ） the rate of blodegradatlon of fluoranthene on the 1st and 6th days （l.e. at 24 and 144 h） was approximately 35% and 85%, respectively; and （Ⅲ） the blodegradatlon capability of C. caspla alga towards fluoranthene was high. It is suggested that further Investigations on the toxicity of fluoranthene towards algae, as well as on algal blodegradatlon mechanisms, are of great Importance to use C. caspla as a biological treatment species In an organic wastewater treatment system.