Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line

Single-walled carbon nanotubes (SWCNT), fullerenes (C₆₀), carbon black (CB), nC₆₀, and quantum dots (QD) have been studied in vitro to determine their toxicity in a number of cell types. Here, we report that classical dye-based assays such as MTT and neutral red (NR) that determine cell viability produce invalid results with some NM (nanomaterials) due to NM/dye interactions and/or NM adsorption of the dye/dye products. In this study, human epidermal keratinocytes (HEK) were exposed in vitro to CB, SWCNT, C₆₀, nC₆₀, and QD to assess viability with calcein AM (CAM), Live/Dead (LD), NR, MTT, Celltiter 96^® AQueous One (96 AQ), alamar Blue (aB), Celltiter-Blue^® (CTB), CytoTox One™ (CTO), and flow cytometry. In addition, trypan blue (TB) was quantitated by light microscopy. Assay linearity (R² value) was determined with HEK plated at concentrations from 0 to 25,000 cells per well in 96-well plates. HEK were treated with serial dilutions of each NM for 24 h and assessed with each of the viability assays. TB, CAM and LD assays, which depend on direct staining of living and/or dead cells, were difficult to interpret due to physical interference of the NM with cells. Results of the dye-based assays varied a great deal, depending on the interactions of the dye/dye product with the carbon nanomaterials (CNM). Results show the optimal high throughput assay for use with carbon and noncarbon NM was 96 AQ. This study shows that, unlike small molecules, CNM interact with assay markers to cause variable results with classical toxicology assays and may not be suitable for assessing nanoparticle cytotoxicity. Therefore, more than one assay may be required when determining nanoparticle toxicity for risk assessment.     

Single walled carbon nanotubes induce indirect cytotoxicity by medium depletion in A549 lung cells

The ability of two types of single walled carbon nanotubes (SWCNT), namely Arc Discharge (AD) and HiPco((R)) single walled carbon nanotubes, to induce an indirect cytotoxicity in A549 lung cells by means of medium depletion was investigated. The nanotubes were dispersed in a commercial cell culture medium and subsequently removed by centrifugation and filtration. Spectroscopic analysis confirmed the removal of the nanotubes and showed differing degrees of alteration of the composition of the medium upon the removal of the nanotubes. The ability to induce an indirect cytotoxic effect by altering the medium was evaluated using two endpoints, namely the Alamar Blue (AB) and the Clonogenic assay. Exposure of the A549 cells to the depleted medium which had previously contained carbonaceous nanoparticles, revealed significant cytotoxicity for both endpoints employed. The results presented demonstrate that single walled carbon nanotubes can induce an indirect cytotoxicity by alteration of cell culture medium (in which they have previously been dispersed) which potentially results in a false positive toxic effect being observed in cytotoxicity studies.     

A new approach to the toxicity testing of carbon-based nanomaterials--the clonogenic assay

The cellular toxicity of three types of carbon nanoparticles, namely HiPco single-walled carbon nanotubes (SWCNT), arc discharge SWCNT and Printex 90 carbon black nanoparticles, was studied on three different cell models including the human alveolar carcinoma epithelial cell line (A549), the normal human bronchial epithelial cell line (BEAS-2B) and the human keratinocyte cell line (HaCaT) using the clonogenic assay. Carbon nanomaterials are known to interact with colorimetric indicator dyes frequently used in cytotoxicity assays. By employing the clonogenic assay, any such interactions could be avoided, allowing a more reliable method for the in vitro toxicity assessment of carbon-based nanoparticles. It could be shown that the toxicity of as produced SWCNT samples differs between cell lines and the SWCNT production method used, with HiPco SWCNT samples being more reactive compared to arc discharge produced SWCNT samples, both eliciting a stronger cytotoxic response than carbon black. Furthermore, it was possible to distinguish between effects on cell viability and cell proliferation by including colony size as an additional endpoint in the clonogenic assay. All three particle types were highly effective in inhibiting cell proliferation in all three cell lines, whereas only HaCaT and BEAS-2B cells also showed decreased cell viability.     

Discriminative cytotoxicity assessment based on various cellular damages

There are several assays currently available for the assessment of cell cytotoxicity, including trypan blue exclusion, lactate dehydrogenase (LDH) release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays. Trypan blue exclusion and LDH release assays are appropriate for evaluating cell membrane damage and a colorimetric MTT assay is available for measuring mitochondrial-related reduction capacity. As these assays were randomly utilized to assess the extent of cell damage, we suggest herein that the assay should be selected in accordance with the prevailing cellular situation. This can be determined by using a variety of cell types with differing reduction status, exogenous and endogenous oxidative stressors, and several different oxidized/reduced molecules. Although the trypan blue exclusion and released LDH assay have proven useful for assessments of necrotic and apoptotic cell death with membrane damage, the LDH assay is not appropriate for the measurement of the number of varied cells without membrane damage. In addition, when the cells were treated with exogenous and endogenous oxidative stressors, MTT reduction was shown to be sensitive to a shift to a more oxidizing cellular environment within a narrow range without loss of membrane integrity, and this effect increased in a linear fashion, dependent on the dosage of cytosolic extracts containing various physiological reductants, small reductive molecules (NADPH and GSH), and artificial DTT reducing agent. Finally, we noted that the MTT assay is available for the determination of small-scale oscillations in cellular reduction status and changes in mitochondrial functional activity, but not for evaluating the cytotoxicity of cells with a higher cellular reduction capacity. Altogether, the findings of this study indicate that tools for the testing of cytotoxicity should be selected differently by considering the correlation between the cellular conditions for various stimuli and the principle underlying the assay system.     

Evaluation of uptake,cytotoxicity and inflammatory effects in respiratory cells exposed to pristine and ‐OH and ‐COOH functionalized multi‐wall carbon nanotubes

Toxic effects were reported for pristine‐multi‐wall carbon nanotubes (p‐MWCNTs) while the role of the functionalization on MWCNT‐induced toxicity is not yet well defined. We evaluated on human alveolar (A549) epithelial cells and normal bronchial (BEAS‐2B) cells exposed to p‐MWCNTs, MWCNTs‐OH and MWCNTs‐COOH: uptake by TEM, cell viability by different assays, membrane damage by the LDH assay and cytokine release by ELISA. The aims of the present study were to: (i) confirm MWCNT cytotoxicity mechanisms hypothesized in our previous studies; (ii) identify the most reliable viability assay to screen MWCNT toxicity; and (iii) to test our model to clarify the role of functionalization on MWCNT‐induced toxicity. In A549 cells, p‐MWCNTs and MWCNTs‐OH were localized free in the cytoplasm and inside vacuoles whereas MWCNTs‐COOH were confined inside filled cytoplasmic vesicles. WST‐1 and Trypan blue assays showed in A549 cells a similar slight viability reduction for all MWCNTs whereas in BEAS‐2B cells WST1 showed a high viability reduction at the highest concentrations, particularly for MWCNTs‐COOH. The MTT assay showed a false cytotoxicity as a result of MWCNTs‐interference. Pristine and MWCNTs‐COOH induced membrane damage, particularly in BEAS‐2B cells. MWCNTs‐COOH induced interleukin‐6 (IL‐6) and IL‐8 release in A549 cells whereas p‐MWCNTs induced IL‐8 release in BEAS‐2B cells. MWCNTs intracellular localization in A549 cells confirms the toxicity mechanisms previously hypothesized, with p‐MWCNTs disrupting the membrane and vesicle‐confined MWCNTs‐COOH inducing inflammation. WST‐1 was more reliable than MTT to test MWCNT‐toxicity. BEAS‐2B cells were more susceptible then A549 cells, particularly to MWCNT‐COOH cytotoxicity. Our results confirm the toxicity of p‐MWCNTs and demonstrate, also for the two kinds of tested functionalized MWCNTs toxic effects with a different mechanism of action. Copyright © 2015 John Wiley & Sons, Ltd.     

Evaluation of eight in vitro assays for assessing the cytotoxicity of cigarette smoke condensate.

The accurate assessment of cytotoxicity is important for evaluating the potential of a test agent to induce pathologies that result from cell killing and to determine appropriate doses for other endpoints, such as genetic toxicology studies. The objective of this work was to determine the most sensitive assays for assessing cytotoxicity in Chinese hamster ovary (CHO) cells following short-term (1 h) and long-term (24 h) exposure to cigarette smoke condensate (CSC). Eight in vitro cytotoxicity assays with different endpoints were used to evaluate the cytotoxicity of Kentucky reference 1R4F (K1R4F) CSC in CHO cells. The assays used for this study were neutral red uptake, LDH release, kenacid blue binding, MTT formation, XTT formation, acid phosphatase activity, sulforhodamine B binding and resazurin binding. Four of the more widely used cytotoxicity assays (neutral red, MTT, kenacid blue and LDH) were also evaluated at 3-, 6-, 12- and 18-h time points. At the 1-h exposure time, LDH was the most sensitive with toxicity observed beginning at 100 microg/ml. None of the other assays demonstrated a concentration-dependent increase in toxicity after 1-h exposures even at the maximum concentration of 150 microg/ml of CSC. Following 24 h of exposure, neutral red and kenacid blue were the most sensitive. The results of our study indicate the assay that measured membrane integrity was the most sensitive for short exposure times, whereas the neutral red and kenacid blue assays that measured total cell number were more sensitive for longer exposure times.     

The MTT assay yields a relatively lower result of growth inhibition than the ATP assay depending on the chemotherapeutic drugs tested.

Accurate assessment of the anti-growth effects of chemotherapeutics is immensely importance in cancer research with regard to drug discovery and toxicological safety. A number of in vitro cytotoxicity assays are used for these purposes. However, there is the possibility for different results in the assessments because the way they measure the viability of cancer cells is specific to each assay. In the present study, the performance of two common assays (MTT and ATP) in the assessment of anti-growth effects of chemotherapeutics on a lung cancer cell line (A549) was compared. The cells were treated with paclitaxel, docetaxel, gemcitabine, 5-fluorouracil (5-FU), etoposide, doxorubicin, epirubicin, cisplatin, 4-hydroperoxycyclophosphamide (4-HC) and carboplatin in six different concentrations. When taking all the drugs and inhibitions into account, a moderate correlation (r=0.670; p=0.01) between the assays was found. However, IC 50 values by the MTT assay were higher in 90% of the drugs than those found by the ATP assay. In addition to this, there was a statistically significant difference between the dose response curves of the assays, which was dependent on the drugs of choice. We recommend caution in comparing these assays to evaluate the anti-growth effects of chemotherapeutics because the MTT assay seem to give rise to relatively lower inhibition (higher viability) levels than the ATP assay, depending on the drugs of choice.     

Cholesterol interferes with the MTT assay in human epithelial-like (A549) and endothelial (HLMVE and HCAE) cells

Metabolically active cells are able to convert the MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye to blue formazan. This is the basis of the MTT assay, which is among the most widely used screening methods to evaluate cell viability and proliferation. When testing the effects of cholesterol products on the viability of human pulmonary epithelial-like A549 cells using trypan blue staining (cell numbers) and the MTT assay, results were inconsistent. The MTT assay indicated greater than 50% loss of viability with exposure of cells to cholesterol, whereas there was no decrease in viability indicated by trypan blue exclusion and propidium iodide uptake. A similar decrease in MTT reduction was obtained upon cholesterol treatment in human lung microvascular endothelial cells (HLMVECs) and human coronary artery endothelial cells (HCAECs) without loss of viability. This suggested a direct interference of cholesterol with the assay. However, using a cell-free system, there was no decrease in the reduction of MTT by ascorbic acid during incubation with a similar concentration of cholesterol. Light microscopy revealed enhanced exocytosis of formazan granules in presence of cholesterol. Incubation with apolipoprotein A-1 decreased cholesterol-mediated inhibition of MTT assay. These studies indicate decreased MTT reduction as a result of enhanced exocytosis of formazan due to cholesterol. A careful validation of viability assay procedures is therefore suggested in experiments where cholesterol is a constituent, to avoid a potential bias in concluding results of cytotoxicity studies.     

In vitro toxicity of serum protein-adsorbed citrate-reduced gold nanoparticles in human lung adenocarcinoma cells

We examined the cytotoxicity effect of the serum protein coated gold nanoparticles (AuNPs) in the A549 cells. Negatively charged AuNPs were prepared by chemical reduction using citrate. The dimension and surface charge of AuNPs were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements. The AuNPs modified by the citrate anion were presumed to adsorb the serum proteins as indicated from the visible absorption spectroscopy, DLS, and quartz crystal microbalance (QCM) data. The QCM results indicated that among the constituents, fetal bovine serum (FBS) should be the major adsorbate species on the AuNPs incubated in the RPMI medium. The internalization of AuNPs into the A549 cells was also monitored using TEM and dark-field microscopy (DFM). Both methylthiazol tetrazolium (MTT) and lactate dehydrogenase (LDH) assays revealed that AuNPs were toxic as determined by their half-maximal inhibitory concentration. A flow cytometric and real-time PCR analysis of apoptotic genes along with the ATP depletion measurements suggested that AuNPs induce cell damages through extrinsic and intrinsic apoptotic pathways.     

Evaluation of the cytotoxicity of cigarette smoke total particulate matter using three in vitro assays and two types of cells

Abstract

In vitro cytotoxicity assays can be used to evaluate potential toxicological effects of tobacco products. Total particulate matter (TPM) from mainstream cigarette smoke trapped by a Cambridge filter is used widely for biological evaluation of smoke. This study compared neutral red uptake (NRU), lactate dehydrogenase (LDH) activity and WST-1 assays for assessing the cytotoxicity of TPM, and evaluated the sensitivity of Chinese hamster ovary (CHO) cells and human lung adenocarcinoma epithelial cell line (A549 cells) to TPM-induced cytotoxic effects. The results indicate that NRU and WST-1 assays are preferable to LDH activity assay for assessing the TPM-induced cytotoxicity, and NRU assay might be more sensitive than WST-1 assay. The cytotoxicity of 3R4F reference cigarettes and two commercial brands of cigarettes were tested by NRU assay in CHO and A549 cells. The results showed that EC₅₀ values in CHO cells treated with TPM were lower than EC₅₀ values in A549 cells, indicating CHO cells are more sensitive to TPM-induced cytotoxic effects than A549 cells.     

In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride

The aim of this study was to compare four in vitro cytotoxicity assays and determine their ability to detect early cytotoxic events. Two hepatoma cell lines, namely HTC and HepG2 cells, were exposed to cadmium chloride (0-300 microM) for 3, 5 and 8 h. Following exposure to the toxic metal cytotoxicity was determined with the lactate dehydrogenase leakage assay (LDH), a protein assay, the neutral red assay and the methyl tetrazolium (MTT) assay. In HTC cells no toxicity was observed for any incubation period when the LDH leakage, the MTT and the protein assay were employed whereas the neutral red assay revealed early cytotoxicity starting after incubation of HTC cells with CdCl(2) for 3 h. In the case of HepG2 cells the MTT assay reveals cytotoxicity due to CdCl(2) exposure after 3 h whereas no such effect is seen with the other three assays. Following 5 h exposure of HepG2 cells to CdCl(2), toxicity is observed with the MTT assay at lower concentrations compared to the ones required for detection of toxicity with the LDH leakage and the neutral red assay. In conclusion different sensitivity was observed for each assay with the neutral red and the MTT assay being the most sensitive in detecting cytotoxic events compared to the LDH leakage and the protein assay.     

多聚二磷酸腺苷核糖聚合酶抑制剂对顺铂在肺癌治疗中增敏的作用研究

目的探讨多聚二磷酸腺苷核糖聚合酶(PARP-1)抑制剂4-氨基-1,8-萘二胺(4-AN)对顺铂在肺腺癌治疗中的增敏作用及相关机制。方法应用MTT法和克隆形成试验检测4-AN与顺铂联合作用对A549细胞的细胞毒性作用;应用单细胞凝胶电泳和微核试验检测4-AN与顺铂联合作用对A549细胞的遗传毒性作用。结果 4-AN可以增加顺铂对A549细胞的杀伤作用,且杀伤作用随4-AN浓度增加而增强;4-AN可以增加顺铂导致的DNA单双链断裂和染色体损伤,而且随药物浓度的增加,损伤作用增强。结论抑制PARP-1可以有效的增加A549对顺铂的敏感性,其作用机制可能是通过抑制A549细胞DNA单双链损伤修复,继而引起染色体损伤,导致细胞的生长和克隆受到抑制。     

Effects of water-soluble functionalized multi-walled carbon nanotubes examined by different cytotoxicity methods in human astrocyte D384 and lung A549 cells

The widespread projected use of functionalized carbon nanotubes (CNTs) makes it important to understand their potential harmful effects. Two cell culture systems, human A549 pneumocytes and D384 astrocytoma cells, were used to assess cytotoxicity of multi-walled CNTs (MWCNTs) with varying degrees of functionalization. Laboratory-made highly functionalized hf-MW-NH₂ and less functionalized CNTs (MW-COOH and MW-NH₂) were tested in comparison with pristine MWCNTs, carbon black (CB) and silica (SiO₂) by MTT assay and calcein/propidium iodide (PI) staining. Purity and physicochemical properties of the test nanomaterials were also determined.     

Comparison of alamar blue and MTT assays for high through-put screening.

The performance of alamar blue and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell viability assays in a high through-put format were compared. A total of 117 drugs chosen for their wide range of therapeutic areas were screened at 10 microM using both assays in human hepatoma cell line HepG2. Except for terfenadine and astemizole, which performed consistently in both assays, the alamar blue assay was slightly more sensitive than the MTT assay for most compounds. The MTT assay was less sensitive detecting an effect for daunorubicin and trifluoperazine. Seven drugs, astemizole, daunorubicin, ellipticine, fluphenazine, terfenadine, thioridazine and trifluoperazine, had percent viability results of 55% or less in the alamar blue assay at the single point screen. These were re-tested in both assays for reconfirmation of cytotoxicity and determination of the EC50 values. Except for daunorubicin, the EC50 values were comparable in both assays. Based on these results and the Z'-factor assessment of assay quality, both assays provided useful information to identify in vitro cytotoxic drugs at early stages of drug candidate selection. However, careful interpretation of data is warranted due to the possibility of false positive or negative results caused by inducers and/or inhibitors of metabolic enzymes that are responsible for transformation of cell toxicity end points, as we demonstrated using dicumarol.     

煤尘对人肺泡上皮细胞DNA损伤作用的体外实验

目的：以体外培养的人肺泡上皮细胞（A549）作为靶细胞，探讨煤尘对DNA的损伤作用。方法：将人肺泡上皮细胞（A549）用3种煤尘作用2h或24h，用MTT法测定细胞存活情况及用单细胞凝胶电泳技术测定DNA链断裂情况；采用两种细胞共培养的方法，用MTT法测定煤尘在巨噬细胞介导作用下对A549细胞存活情况的影响及用单细胞凝胶电泳技术测定DNA链断裂情况。结果：3种煤尘（游离SiO2含量分别为4．33％、1．79％、0．67％）以3种剂量（100、500、1000μg/ml）直接作用于A549细胞24h，在500和1000μg/ml时均可引起细胞存活率的明显下降；直接作用于A549细胞2h或24h及通过巨噬细胞介导作用下A549细胞，均未能引起A549细胞DNA链断裂程度的明显增加。结论：煤尘对A549细胞具有明显的细胞毒性，但不具有明显的遗传毒性。     

Modulation of dipyridamole action by alpha 1 acid glycoprotein. Reduced potentiation of quinazoline antifolate (CB3717) cytotoxicity by dipyridamole

Dipyridamole potentiates the cytotoxicity of N10-propargyl-5,8-dideazafolic acid (CB3717), an antifolate inhibitor of thymidylate synthase, by inhibiting both thymidine (TdR) salvage and deoxyuridine (UdR) efflux. Dipyridamole binds to the serum component alpha 1acid glycoprotein (alpha 1AGP) and hence the effects of alpha 1AGP on dipyridamole-induced changes in nucleoside transport and CB3717 cytotoxicity have been investigated. Using A549 lung cancer cells in vitro, alpha 1AGP reduced the inhibition of nucleoside transport by dipyridamole in a concentration-dependent manner. Between 10 and 200 times the concentration of dipyridamole was needed to inhibit TdR uptake to the same degree in medium containing 1 mg/ml alpha 1AGP (a physiological concentration) when compared to the uptake in alpha 1AGP-free medium. Although dipyridamole inhibited UdR efflux more than TdR efflux, inhibition of UdR efflux was reduced less than the inhibition of TdR efflux in the presence of 1 mg/ml alpha 1AGP. Thus, clinically achievable levels of dipyridamole (2.5-7.5 microM), even in the presence of physiological alpha 1AGP concentrations, caused significant inhibition of nucleotide uptake and efflux. The cytotoxicity of CB3717 was increased 2-3-fold by 3 and 10 microM dipyridamole in alpha 1AGP-free medium, whereas dipyridamole did not significantly (P greater than or equal to 0.05) potentiate CB3717 cytotoxicity in the presence of 1 mg/ml alpha 1AGP. Measured free dipyridamole levels indicated that the impaired inhibition of nucleoside transport and the lack of potentiation of CB3717 cytotoxicity in the presence of alpha 1AGP was due solely to the binding of dipyridamole to alpha 1AGP. It is concluded that alpha 1AGP levels will be a major determinant of the ability of dipyridamole to modulate the activity of antimetabolites in vivo.     

Evaluation of MTT assay for measurement of emodin-induced cytotoxicity

The specificity and sensitivity of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium assay can be influenced by certain factors, such as colored substances and cell volume. When the MTT assay is applied to measure cytotoxicity induced by emodin, its accuracy might be affected by emodin itself. Vascular smooth muscle cells were cultured in M199 medium. The optical density of emodin or formazan was measured by spectrophotometry. Emodin has a different absorption spectrum in different solvents. The solvents containing water induced a red shift of the absorption curve of emodin, which increased the overlap of the absorption curves of emodin and formazan. Formazan was formed from the MTT tetrazolium salt by emodin in a dose-dependent manner, which was partially suppressed by serum. Cytotoxicity was induced by emodin in a time- and dose-dependent manner in a modified MTT assay. The data suggest that emodin can alter the accuracy of the MTT assay but that a modified MTT assay is still valuable in measuring emodin-induced cytotoxicity.     

Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells

IntroductionPrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity.Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts.MethodsThe anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1–200 μg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol — a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB).ResultsAt higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC₅₀ values compared to other modes of measurement, however, the statistically significant differences in EC₅₀ values among the assays were revealed only for spent hop extract. Conversely, the EC₅₀ values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC₅₀ values, the cytotoxicity of plant compounds ranked as follows: spent hop extract > resveratrol > walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay.DiscussionIn conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC₅₀ values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided.     

Can in-vitro assays predict chemically-induced skin damage?

Eleven chemicals with a known potential to damage skin as corrosive, irritants or non-irritants, have been tested for their ability to affect the function and viability of mammalian cells in three in vitro assays. The assays measured total protein content by Coomassie Blue R binding (CB), lysosomal integrity by Neutral Red uptake (NR), or mitochondrial function by MTT uptake/reduction. Two cell lines, Chinese Hamster Ovary cells (CHO) and Swiss Albino Mouse Fibroblasts (3T3), were compared. The results of all three assays agreed well with one another and with the observed number and morphology of the cells for each chemical tested. The CB tended to be the least sensitive and the NR the most variable of the three assays. Overall the MTT assay was most reliable, but may give anomalous results if used alone. CHO and 3T3 cells gave similar results, although 3T3 cells tended to be less sensitive than CHO cells. This may depend principally on the rate of division and proportion of dividing cells. Using the results from the MTT assay, corrosive chemicals were cytotoxic, since concentrations of less than 10 μg/ml reduced dye uptake by 50%. Chemicals which were skin irritants were moderately cytotoxic, since concentrations in the order of 50–500 μg/ml reduced dye uptake by 50%. Non-irritant chemicals were not cytotoxic up to 5 mg/ml. In decreasing order of cytotoxicity, the chemicals were ranked: tributyltin chloride, dibutyltin dichloride, silver nitrate, benzalkonium chloride, zinc monoglycerolate, sodium dodecyl sulphate, benoxaprofen, fenclofenac, n-hexane, butan-1-ol, 2-methoxyethanol. The categorization and ranking of the degree of skin irritation concurred with the degree of cell kill. These results indicate that such simple in-vitro tests could be used instead of animals to predict skin irritants at an early stage of hazard identification.     

氟尿嘧啶磁性丝心蛋白-壳聚糖微球体外对癌细胞的毒性作用

采用改良的MTT比色法测定不同培育时间和浓度下,氟尿嘧啶(1)磁性丝心蛋白-壳聚糖微球(magnetic fibroinchitosan microspheres,MFCMs)在体外对人肝癌细胞株SMMC-7721和人肺腺癌细胞株A549生长的抑制作用.结果表明,1 MFCMs对SMMC-7721的抑制表现为明显的时效和量效特征;对A549的抑制主要呈时效关系.由半数有效浓度(IC50)可知,1 MFCMs对A549比SMMC-7721更敏感.1 MFCMs与1注射剂对两种癌细胞的抑制率有显著差异(P＜0.05).