Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential

While drug toxicity (especially hepatotoxicity) is the most frequent reason cited for withdrawal of an approved drug, no simple solution exists to adequately predict such adverse events. Simple cytotoxicity assays in HepG2 cells are relatively insensitive to human hepatotoxic drugs in a retrospective analysis of marketed pharmaceuticals. In comparison, a panel of pre-lethal mechanistic cellular assays hold the promise to deliver a more sensitive approach to detect endpoint-specific drug toxicities. The panel of assays covered by this review includes steatosis, cholestasis, phospholipidosis, reactive intermediates, mitochondria membrane function, oxidative stress, and drug interactions. In addition, the use of metabolically competent cells or the introduction of major human hepatocytes in these in vitro studies allow a more complete picture of potential drug side effect. Since inter-individual therapeutic index (TI) may differ from patient to patient, the rational use of one or more of these cellular assay and targeted in vivo exposure data may allow pharmaceutical scientists to select drug candidates with a higher TI potential in the drug discovery phase.     

Osmotic microenvironments within tissue culture well plates are an important cause of variability in plant protoplast cultures

Summary To determine if the medium osmolality of plant protoplast cultures in 24-well tissue culture plates changes sufficiently during the culture period to affect development of the protoplasts the osmolality and the division of the protoplasts was monitored within the plates over a three week period. Large increases in osmolality were measured in cell-free experiments indicating that overall evaporation from the plates was substantial. The amount of evaporation from a given culture well depended on the position of the well within the plate; three microenvironments corresponding to the corner, edge, and middle positions of the plate consistently developed. Water placed in the recessed area between each culture well moderated the desiccation of the medium but did not eliminate the formation of microenvironments. The osmolality of the medium in protoplast cultures was higher than in the cell-free experiments but similar trends in terms of plate position were recorded. After 3 weeks of incubation of plates with water added between the wells, the osmolality of the medium in the protoplast cultures had increased 209 mOsm in the corner wells, had increased 77 mOsm in the edge wells, and had decreased 39 mOsm in the middle wells. As a result, there was a three-fold higher incidence of division of the protoplasts in the middle wells than in the corner wells. The non-uniformity of medium osmolality in protoplast cultures within tissue cultures well plates is important in experimental design, in the reproducibility of procedures between different laboratories, and in the preparation of replenishment medium for protoplast cultures to minimize osmotic shock. This work was supported by the College of Agriculture and Life Sciences and the Graduate School, University of Wisconsin-Madison; by McIntire-Stennis project WIS 3082; and by the USDA-Forest Service, North Central Forest Experiment Station (B. Haissig, Project Leader).     

广西灵香草挥发油化学成分

灵香草(Lysimachia foenum-graecum Hance)又名零陵香,为报春花科(Primulaceae)排草属(Lysimachia L.)植物,主产广西、云南、广东、四川、贵州等地[1].灵香草性味甘、淡、平,具行气、止痛、驱蛔功能,药理实验证明具有抑制流感病毒作用[2]; 其挥发油香气浓郁且持久,是烟用香精的重要赋香剂之一.     

ACAT1 deletion in murine macrophages associated with cytotoxicity and decreased expression of collagen type 3A1

In contrast to some published studies of murine macrophages, we previously showed that ACAT inhibitors appeared to be anti-atherogenic in primary human macrophages in that they decreased foam cell formation without inducing cytotoxicity. Herein, we examined foam cell formation and cytotoxicity in murine ACAT1 knockout (KO) macrophages in an attempt to resolve the discrepancies. Elicited peritoneal macrophages from normal C57BL6 and ACAT1 KO mice were incubated with DMEM containing acetylated LDL (acLDL, 100 microg protein/ml) for 48h. Cells became cholesterol enriched and there were no differences in the total cholesterol mass. Esterified cholesterol mass was lower in ACAT1 KO foam cells compared to normal macrophages (p<0.04). Cytotoxicity, as measured by the cellular release of [(14)C]adenine from macrophages, was approximately 2-fold greater in ACAT1 KO macrophages as compared to normal macrophages (p<0.0001), and this was independent of cholesterol enrichment. cDNA microarray analysis showed that ACAT1 KO macrophages expressed substantially less collagen type 3A1 (26-fold), which was confirmed by RT-PCR. Total collagen content was also significantly reduced (57%) in lung homogenates isolated from ACAT1 KO mice (p<0.02). Thus, ACAT1 KO macrophages show biochemical changes consistent with increased cytotoxicity and also a novel association with decreased expression of collagen type 3A1.     

Assessment of environmental contamination associated with a mammalian cell transformation assay

Summary To estimate worker exposures to, and environmental contamination from, test chemicals and organic solvents used in an in vitro assay to assess the carcinogenic potential of chemicals, sodium fluorescein, a noncarcinogenic fluorescent material, was dissolved in tissue culture medium used to maintain early passage hamster embryo cells. Personal and environmental samples were taken over a 14-d period. The assay was performed according to standard procedures in a ventilated glove box or laminar flow safety cabinet. Considerably more than 99% of the chemical contamination found was recovered from the interiors of the glove box and hood and from disposable equipment. Contamination outside the containment units (less than 1 μg) resulted from intralaboratory transport of chemicals, treated cultures, and contaminated equipment. We conclude that the standard operating practices and procedures provided adequate safeguards for personnel and the environment. Research sponsored by the National Cancer Institute under Contract N01-CO-75380, with Litton Bionetics, Inc.     

福建石荠荣挥发油化学成分分析

报道了福建省不同产地石蔼荣全草挥发油含量在不同生育期的变化,其含量变化范围为0.320%-1.020%。对五个不同产地的全草挥发油主要化学成分进行分析,其挥发油主要成分为：番荆芥酚、1,8－桉叶油素、百里香酚、β－石竹烯、侧柏酮、异胡萍荷酮等,不同产地的挥发油化学成分存在遗传相似性,但亦有一定差异,石荠荣资源在香精香料、医药、化妆品等行业具有广阔的开发利用前景。     

Increased resistance to nitric oxide cytotoxicity associated with differentiation of neuroblastoma-glioma hybrid (NG108-15) cells

Nitric oxide (NO), synthesized by the enzyme nitric oxide synthase (NOS), acts as an intercellular messenger associated with various physiological and pathological events. In this study, we investigated whether there exits a difference in the vulnerability to NO-induced cytotoxicity between undifferentiated and differentiated NG108-15 cells, and if so, the mechanisms responsible for the difference. Following a 7- to 8-day exposure to dibutyryl cAMP (dbcAMP), NG108-15 cells exhibited a neuron-like morphology associated with the expression of the neuronal protein, synaptophysin, and with increased NADPH-d activity. Neuron-like differentiated NG108-15 cells acquired resistance to exogenously applied NO. This increased resistance to NO toxicity in differentiated cells was almost completely cancelled out by inhibiting the activity of superoxide dismutase (SOD), but not by inhibiting the activity of NOS. The present study suggested that the activity of SOD increased in parallel with the activity of NOS associated with differentiation and was crucial for the acquired resistance to NO toxicity in differentiated cells.     

Capture of volatile metabolites for tracking the evolution of gibberellic acid in a solid-state culture of Gibberella fujikuroi

The evolution of volatile compounds produced during solid substrate cultivation (SSC) of Gibberella fujikuroi on wheat bran was tracked looking for volatile metabolites related with GA₃ production. Ethyl acetate and isoamyl acetate gave identical profiles and sharp increases that abated as the production of GA₃ began, while ent-kaurene displayed a profile matching that of the development of GA₃. ent-Kaurene is a precursor in the synthesis of gibberellins and was the most abundant compound found.     

Evaluation of the proposed interaction of nucleic acid with Clostridium difficile toxins A and B and the effects of nucleases on cytotoxicity

Both DNA and RNA were found to co-purify with Clostridium difficile toxin B but not toxin A. DNAase treatment greatly reduced the cytotoxicity of toxin B but not of toxin A. RNAase had no effect on either toxin. The effects on toxin B were shown to be due to a contaminating protease and could be inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride.     

A quantitative assessment of the cytotoxicity associated with chromosomal aberration detection in Chinese hamster ovary cells.

Regulatory guidelines suggest testing chemicals up to cytotoxic doses in chromosomal-aberration assays. To investigate the utility and limitations of various cytotoxicity indicators we used Chinese hamster ovary (CHO) cells to test 8 chemicals with differing ratios of cytotoxicity to clastogenicity. We measured immediate or delayed cell killing and growth inhibition (ATP levels, cell counts, colony-forming efficiency, CFE) and cell-cycle perturbations (mitotic index, MI; average generation time, AGT). Aberrations (abs) were scored 10 and 24 h from the beginning of the 3-h treatment. All 8 compounds induced abs at concentrations that reduced cell growth at 24 h by 50% or less. Concentrations of each chemical which induced at least 15% cells with abs, gave little loss of CFE (0-20%) for mitomycin C, adriamycin, cadmium sulfate and 2,6-diaminotoluene in contrast to the marked loss of CFE (70-80%) for eugenol (EUG), 2-aminobiphenyl and 8-hydroxyquinoline (8-HQ). 2,4-Diaminotoluene (2,4-DAT) was intermediate. Higher aberration yields were found at 24 h than at 10 h, even when minimal cell-cycle delay was detected by AGT estimates from BrdUrd-labeled cells. Cells with multiple abs were seen at 24 but not at 10 h, and often confirmed clastogenicity when there was only a weak increase in the percentage of cells with aberrations. Total ATP per culture did not always correlate with cell number, especially at later times after treatment. This is likely due to metabolic perturbations or altered cell biomass that are known to affect cell ATP content. MI suppression often did not correlate with AGT, e.g., only small increases in AGT were seen for 8-HQ, 2,4-DAT and EUG despite severe mitotic suppression at 10 h. By 24 h the MI for all chemicals had recovered, sometimes exceeding control levels. Marked mitotic accumulation was seen at 10 h for 2,4-DAT, indicating cell synchrony. Thus, the MI has limited value for dose selection. In conclusion, even weakly active chemicals were detected at a single time without exceeding a 50% growth reduction at 24 h.     

红豆树叶挥发油化学成分及其抗氧化和抑菌活性研究

为探究红豆树叶挥发油的化学组成及其生物活性,本研究首次采用水蒸气蒸馏法提取红豆树叶挥发油,并通过气相色谱-质谱联用技术(GC-MS)分析其化学成分,结合DPPH和ABTS法及抑菌圈法,评价其体外抗氧化和抑菌活性。结果表明,从红豆树叶挥发油中共检测出化合物36个,占挥发油总量的90.50%;挥发油主要成分为1,4-二十烷二烯(25.72%)、1,19-二十烷二烯(10.85%)、2,6-二叔丁基对甲酚(10.14%)、邻苯二甲酸正丁基异丁基酯(9.75%)、(Z,Z)-6,9-二十烷二烯(7.60%)、(E,E)-α-金合欢烯(7.51%)、叶醇(4.74%)和2-异丙烯基-5-甲基-6-庚烯-1-醇(4.04%)。红豆树叶挥发油对DPPH自由基和ABTS自由基清除作用的半数有效量(ED₅₀)分别为0.27、0.14 mg/mL,且抗氧化活性与挥发油浓度呈量效相关。红豆树叶挥发油浓度为7.1 mg/mL时,其对枯草芽孢杆菌(Bacillus subtilis)、金黄色葡萄球菌(Staphylococcus aureus)、绿脓杆菌(Pseudomonas aeruginosa)和大肠杆菌(Escherichia coli)的抑菌圈分别为11.29、9.88、10.85和11.03 mm。本研究为红豆树叶资源的综合开发利用提供了理论基础。     

Sequence specificities in the interactions of chemicals and radiations with DNA

Summary Sequence specificities in the interactions of chemicals and radiations with DNA are reviewed. Emphasis is placed on information which has been obtained by adapting DNA sequencing techniques to the detection of DNA damage and modifications. The actions of anti-tumor drugs, non-covalent DNA binders and UV irradiation are discussed in terms of both modifications induced in DNA and the subsequent response of DNA polymerase and repair enzymes. Particular attention is paid to the evidence for sequence-specific interactions of these agents with DNA. It is concluded that while most agents exhibit warm or even hot spots in their interactions with DNA there is not, as yet, compelling evidence for extreme selectivity down to say the gene level in their actions. There does, however, appear to be some affinity, particularly in the case of non-covalent binders, for certain tertiary structures rather than primary sequences per se. In addition, both misincorporation opposite DNA damage and the bypass of damage by polymerases are important phenomena which, to some extent, exhibit sequence specificities. The idea is advanced that DNA structures such as hairpins or cruciforms maybe important in vivo targets for many agents giving rise to specific biological effects.     

Study of the cytotoxicity and particle action in human cancer cells of titanocene-functionalized materials with potential application against tumors

Titanocene dichloride [Ti(η⁵-C₅H₅)₂Cl₂] (1), has been grafted onto dehydrated hydroxyapatite (HAP), Al₂O₃ and two mesoporous silicas MSU-2 (Michigan State University Silica type 2) and HMS (Hexagonal Mesoporous Silica), to give the novel materials HAP/[Ti(η⁵-C₅H₅)₂Cl₂] (S1) (1.01 wt.% Ti), Al₂O₃/[Ti(η⁵-C₅H₅)₂Cl₂] (S2) (2.36 wt.% Ti), HMS/[Ti(η⁵-C₅H₅)₂Cl₂] (S3) (0.75 wt.% Ti) and MSU-2/[Ti(η⁵-C₅H₅)₂Cl₂] (S4) (0.74 wt.% Ti), which have been characterized by powder X-ray diffraction, X-ray fluorescence, nitrogen gas sorption, multinuclear magic angle spinning NMR spectroscopy, IR spectroscopy, thermogravimetry analysis, UV spectroscopy, scanning electronic microscopy and transmission electronic microscopy. The cytotoxicity of the titanocene-functionalized materials toward human cancer cell lines from five different histogenic origins: 8505 C (anaplastic thyroid cancer), A253 (head and neck cancer), A549 (lung carcinoma), A2780 (ovarian cancer) and DLD-1 (colon cancer) has been determined. M₅₀ values (quantity of material needed to inhibit normal cell growth by 50%) and Ti-M₅₀ values (quantity of anchored titanium needed to inhibit normal cell growth by 50%) indicate that the activity of S1-S4 against studied human cancer cells depended on the surface type as well as on the cell line. In addition, studies on the titanocene release and the interaction of the materials S1-S4 with DNA show that the cytotoxic activity may be due to particle action, because no release of titanium complexes has been observed in physiological conditions, while electrostatic interactions of titanocene-functionalized particles with DNA have been observed.     

Combined use of six-well polystyrene plates and thin layer chromatography for rapid development of optimal plant cell culture processes: application to taxane production byTaxus sp.

TwoTaxus (T. chinensis andT. baccata) cell suspension cultures were used as a model system to demonstrate the similarities of biomass accumulation and secondary metabolite (taxane) production obtained from cultures in six-well polystyrene plates and glass shake flasks (25 ml and 125 ml). Interference from binding of taxanes in cell-free culture broth to the polystyrene plates was minimal with 85% of the paclitaxel (Taxol®) and 100% of baccatin and 10-deacetyl-7-xylosyl-taxol remaining in the medium after 24 h beyond which no further binding was observed. A simple thin layer chromatography (TLC) procedure with a chloroform: acentonitrile (4:1) solvent system on silica gel was developed to simultaneously test up to 17 cultures for taxane production. The combination of six-well plate technology for experimentation and TLC for rapid taxane analysis can greatly accelerate the establishment of conditions for an optimalTaxus plant-cell culture process for taxane production.Abbreviations TLC Thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - HPLC high pressure liquid chromotography - UV ultraviolet - Rf retention factor     

Investigating the lysis of small-cell lung cancer cell lines by activated natural killer (NK) cells with a fluorometric assay for NK-cell-mediated cytotoxicity

Activation of natural killer (NK) cells with interleukin-2 (IL-2) and IL-12 leads to an enhanced lysis of tumour cells. We investigated the ability of NK cells, with or without prior activation, to lyse a variety of small-cell lung cancer (SCLC) target cells. Specific lysis was measured with a fluorometric assay for NK-cell-mediated cytotoxicity: target cells were labelled with 3,3′-dioctadecyloxacarbocyanine, a green membrane dye. After co-incubation with NK cells, dead target cells were stained with propidium iodide, a red DNA dye that only penetrates dead cells. Of all eight SCLC cell lines tested, three were susceptible to lysis by non-activated NK cells, three were only susceptible to lysis by NK cells activated with IL-2 and IL-12 and two were not even susceptible to lysis by activated NK cells. The differences in target cell susceptibility showed no correlation with the expression of MHC-I on the surface of the target cells or with the expression of the adhesion molecules CD50, CD54, CD58 or CD102. Comparing the kinetics of the lysis of one SCLC cell line sensitive to non-activated NK cells and one sensitive only to activated NK cells, we found that maximum lysis of the former was obtained after 1 h, whereas significant lysis of the latter was only obtained after 4 h of incubation. This might be due to different mechanisms engaged in target cell lysis. Received: 23 December 1998 / Accepted: 8 April 1999     

收集挥发性样品的吸附剂法和固相微萃取法

本文介绍挥发性信息化学物质的两种收集方法——吸附剂收集法和固相微萃取收集法,以植物挥发性化学物质收集为例为例,详述了两种方法的原理、步骤、注意事项和实例,最后比较了两种收集方法的优缺点,供相关工作者使用时参考。     

A model culture system with human gingival fibroblasts for evaluating the cytotoxicity of dental materials

Summary A model experimental culture system and protocol are described to screen polymerized dental materials for diffusible toxic products. The system employs cultures of human gingival fibroblasts grown in plates containing immobilized samples of polymerized resins. Comparative cytotoxicity is evaluated by counting viable cells with the aid of phase optics at several time periods up to 48 h. To achieve adequate statistical sampling, multiple counts are made in four different zones at 90° angles from each sample and at three distances from the centers of samples. The most significant data were generated during a 24 to 48 h test period in culture. This cytotoxicity test measured cell death as a function of time of exposure and distance from the sample (24 h, 0 to 3 mm; 48 h, 3 to 6 mm) and permitted a calculation of the relative cytotoxicity for each material, which is termed the viability index (VI). This can be expressed as a percentage related to the control, which is called the time-distance cytotoxicity index (TDCI). This method is simple to carry out because it uses basic laboratory equipment, is rapid, and has a sound scientific basis. It focuses on times and distances when or where, or both, the greatest cellular changes are taking place. Some data illustrated are based on the screening of eight different restorative resins. The literature of cell culture testing of dental materials is reviewed. It is concluded that biotoxicity studies ideally should employ diploid human target cells from the oral cavity because the cells retain specialized features. Secondary cultures or strains of human diploid gingival fibroblasts, which are relatively easy to obtain and maintain, are recommended as cells of choice for screening dental restorative materials in vitro. This project was supported in part by an intramural grant from the Louisiana State University School of Dentistry, derived from BRSG Grant S07-RR-05704-10 awarded by the Biomedical Research Grant Program, Division of Research Resources, National Institutes of Health, Bethesda, MD.