Acute cytotoxicity testing with cultured human lung and dermal cells

Summary An extensive in vitro study with cultured cells was conducted to test the basal cytotoxicity theory. This theory suggests that most chemical injury, at least in vitro, is a manifestation of one or more insults to the basic cellular structures and functions common to mammalian cells. This accounts for the similarity of results in multilaboratory studies. Human fetal lung fibroblasts (HFL1), and human skin fibroblasts (WS1, Detroit551) were studied in culture to evaluate their potential to screen for cytotoxicity. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 h, and the MTT assay was used to assess toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Twenty-nine chemicals were tested with each cell line and the cytotoxicity data compared to rodent and human lethal concentrations. The data suggest that the experimental IC₅₀ values are as accurate predictors of human toxicity as equivalent toxic blood concentrations derived from rodent LD₅₀s. In addition, lung and skin fibroblasts revealed no significant differences among the three cell lines. The results support the conclusion that finite cell lines of human origin have the potential for screening chemicals for human toxicity. In combination with previously published reports, the data suggest that a basal cytotoxic phenomenon may explain the similarity of results among different human cell lines.     

Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay

A colorimetric MTT (tetrazolium salt) cleavage test was used to evaluate cytotoxicity of twenty-three Fusarium mycotoxins on two cultured human cell lines (K-562 and MIN-GL1) as well as their inhibitory effect on proliferation of phytohemagglutinin-stimulated human peripheral blood lymphocytes. The values of 50% inhibition of lymphocyte blastogenesis were very close to the 50% cytotoxic doses observed with the more sensitive cell line (MIN-GL1). T-2 toxin was the most cytotoxic with CD₅₀ and ID₅₀ values less than 1 ng/ml. Type A trichothecenes were the most cytotoxic followed by the type B trichothecenes; the non-trichothecenes were the least cytotoxic. The MTT cleavage test, in conjunction with cell culture, is a simple and rapid bioassay to evaluate cytotoxicity and immunotoxicity of Fusarium mycotoxins.Abbreviations Ac acetyl - ACU acuminatin - DAS diacetoxyscirpenol - DON deoxynivalenol - FUS fusarenon-X - HT-2 HT-2 toxin - MC mononuclear cell - MTT 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide - NEO neosolaniol - NIV nivalenol - NT-1 4,8-diacetoxy T-2 tetraol - PBS phosphate buffered saline - TAT-2T tetraacetoxy T-2 tetraol - T-2 T-2 toxin     

Antifungal and antibacterial activities of indigenous <Emphasis Type="Italic">Streptomyces</Emphasis> isolates from saline farmlands: prescreening,ribotyping and metabolic diversity

A culture collection of 110 indigenous Streptomyces strains originally isolated from saline farmlands (Punjab, Pakistan) using stringent methods was screened biologically and chemically to investigate their potential for the production of bioactive secondary metabolites. In a biological screening the crude extracts obtained from the culture broth of selected strains were analysed for their activity against a set of test organisms, including Gram-positive, Gram-negative bacteria, fungi and microalgae using the disk diffusion bioassay method. Additionally a cytotoxicity test was performed by means of the brine shrimp microwell cytotoxicity assay. In a chemical screening each of the crude extracts was analysed by TLC using various staining reagents and by HPLC-MS/MS measurements. The results depicted an impressive chemical diversity of crude extracts produced by these strains. The taxonomic status of the selected strains was confirmed by preliminary physiological testing and 16S rRNA gene sequencing.     

Toxicity Screening of a Combinatorial Library: Correlation of Cytotoxicity and Gene Induction to Compound Structure

Combinatorial chemistry has increased the number of compounds available for efficacy and safety assessment by several orders of magnitude and has made high throughput assays essential. To test whether higher throughput toxicity assays could be of utility in screening compounds in early development, a selected set of combinatorial chemistry compounds was screened for induction of 70-Kd heat shock protein (HSP70) and 45-Kd growth arrest and DNA damage protein (GADD45) mRNA levels as well as cytotoxicity, in HepG2 cells, using a 96-well microtiter plate format. Both assays, the branched DNA (Quantigene) assay for mRNA levels and MTT for cytotoxicity, were robust enough to be incorporated into a screening format using a single replicate and a single concentration of compound. Significantly, a structure/toxicity correlation was established with this set of compounds with cytotoxicity and gene induction patterns linked to compound structure. Therefore, this type of early screening may be useful in identifying toxic substituents, enabling the design of libraries with less potential for toxicity. While structure/toxicity correlations were observed, no relationship was observed between GADD45 gene induction and mutagenesis as measured by the Ames bacterial reverse mutation assay.     

NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

MTT reduction is usually analysed by colorimetric assay to study mitochondrial dehydrogenase activity as a test of cytotoxicity. This enzymatic reaction produces dark-blue granules of formazan, which increase cell refringency. In this work, we define the conditions for MTT use in quantitative flow cytometric analysis. MTT reduction provides a non-fluorescent dye usable by this technique to study an intracellular NADH-dependent dehydrogenase activity in vital cells. We observe that formazan production increases asymptotically with cell concentration and that this temperature-dependent Michaelis enzymatic reduction is produced essentially by mitochondrial dehydrogenases. In isolated mitochondria from rat hepatocytes and in whole L1210 murine leukemia cells, the Michaelis constants (KM) observed in the presence of respiratory substrates were, respectively, 10 microM and 500 microM. The inhibition of mitochondrial protein synthesis by chloramphenicol, which induces a rise of MTT reduction due to the correlative stimulation of glycolysis (Pasteur effect), is a limit of the MTT assay as a cytotoxicity test.     

Development of an <Emphasis Type="Italic">in vitro</Emphasis> assay for the investigation of metabolism-induced drug hepatotoxicity

In a number of adverse drug reactions leading to hepatotoxicity drug metabolism is thought to be involved by generation of reactive metabolites from nontoxic drugs. In this study, an in vitro assay was developed for measurement of the impact of metabolic activation of compound on the cytotoxicity toward a human hepatic cell line. HepG2 cells were treated for 6 h with compound in the presence or absence of rat liver S9-mix, and the viability was measured using the MTT test. The cytotoxicity of cyclophosphamide was substantially increased by S9-mix in the presence of NADPH. Three NADPH sources were tested: NADPH (1 mmol/L) or NADPH regenerating system with either NADP⁺/glucose 6-phosphate (G6P) or NADP⁺/isocitrate. All three NADPH sources increased the cytotoxicity of cyclophosphamide to a similar extent. Eight test compounds known to cause hepatotoxicity were tested. For these, only the cytotoxicity of diclofenac was increased by S9 enzymes when an NADPH regenerating system was used. The increased toxicity was NADPH dependent. Reactive drug metabolites of diclofenac, formed by NADPH-dependent metabolism, were identified by LC-MS. Furthermore, an increase in toxicity, not related to enzymatic activity but to G6P, was observed for diclofenac and minocycline. Tacrine and amodiaquine displayed decreased toxicity with S9-mix, and carbamazepine, phenytoin, bromfenac and troglitazone were nontoxic at all tested concentrations, with or without S9-mix. The results show that this method, with measurement of the cytotoxicity of a compound in the presence of an extracellular metabolizing system, may be useful in the study of cytotoxicity of drug metabolites.     

The cytotoxic effect of fumonisin B<Subscript>1</Subscript> and ochratoxin A on human and pig lymphocytes using the Methyl Thiazol Tetrazolium (MTT) assay

Lymphocytes cell obtained from healthy human donors and pigs were exposed to fumonisin B₁ (FB₁) and ochratoxin A (OTA), which have been found to be immunosuppressive, carcinogenic and mutagenic, to ascertain their single and combined cytotoxic effects with time and to assess the suitability of animal lymphocytes as test agents in comparison to human cells. The main objectives of this work were to assess the use of animal lymphocytes, particularly pig lymphocytes, for their use in the Methyl Thiazol Tetrazolium (MTT) cytotoxicity test, making them more accessible to animal research-based institutes in comparison to human lymphocytes previously used, and to study the cytotoxic and synergism or antagonistic effects of FB1 and OTA. The MTT assay, which measures cell viability and proliferation based on reduction of MTT to a blue dye, also used the addition of phytohaemagglutinin (PHA) to stimulate the blood cells. The results showed a progressive decrease in lymphocytes viability with time of exposure to the toxins. It was also noted that FB1, as compared to OTA, had a lower cytotoxicity on both human and pig lymphocytes cells. In addition, when the two mycotoxins were combined, a synergistic decrease of cell viability in both human and pig lymphocytes was observed, with pig lymphocytes showing a greater sensitivity. This study has shown that the MTT assay can be used for the determination of cytotoxicity of mycotoxins using animal, and in particular pig, lymphocytes, which eliminates the use of human donors and other cell cultures.     

Design,Synthesis and in Vitro Tumor Cytotoxicity Evaluation of 3,5‐Diamino‐N‐substituted Benzamide Derivatives as Novel GSK‐3β Small Molecule Inhibitors

Glycogen synthase kinase‐3 (GSK‐3) plays an important regulatory role in various signaling pathways; such as PI3 K/AKT, which is closely related to the occurrence and development of tumors. At present, the most reported active GSK‐3 inhibitors have the same structure: lactam ring or amide structure. To find out the GSK‐3β small molecule inhibitor with novel, safe, efficient and more uncomplicated synthesis method, we analyzed in‐depth reported crystal‐binding patterns of GSK‐3β small molecule inhibitor with GSK‐3β protein, and designed and synthesized 17 non‐reported 3,5‐diamino‐N‐substituted benzamide compounds. Their structures were confirmed by ¹H‐NMR, ¹³C‐NMR, and HR‐MS. The preliminary screening of tumor cytotoxicity of compounds in vitro was detected by MTT, and their structure–activity relationships were illustrated. The results have shown that 3,5‐diamino‐N‐[3‐(trifluoromethyl)phenyl]benzamide ( 4d ) exhibited significant tumor cytotoxicity against human colon cancer cells (HCT‐116) with IC₅₀ of 8.3 μm and showed commendable selectivity to GSK‐3β. In addition, Compound 4d induced apoptosis to some extent and possessed modest PK properties.     

Differential effect of ascorbic acid and n-acetyl-L-cysteine on arsenic trioxide-mediated oxidative stress in human leukemia (HL-60) cells

Arsenic trioxide (ATO) has been recommended for the treatment of refractory cases of acute promyelocytic leukemia (APL). Recent studies in our laboratory indicated that oxidative stress plays a key role in ATO-induced cytotoxicity in human leukemia (HL-60) cells. In the present investigation, we performed the MTT assay and trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test to determine the levels of malondialdehyde (MDA) production in HL-60 cells coexposed to either ascorbic acid (AA) and ATO or to n-acetyl-L-cysteine (NAC) and ATO. The results of MTT assay indicated that AA exposure potentiates the cytotoxicity of ATO in HL-60 cells, as evidenced by a gradual increase in MDA levels with increasing doses of AA. In contrary, the addition of NAC to ATO-treated HL-60 cells resulted in a dose-dependent decrease of MDA production. From these results, we conclude that the addition of the AA to ATO-treated HL-60 cells enhances the formation of reactive oxygen species (ROS), whereas the addition of NAC under the same experimental condition significantly (p < .05) decreases the level of ROS formation. On the basis of these direct in vitro findings, our studies provide evidence that AA may extend the therapeutic spectrum of ATO. The coadministration of NAC with ATO shows a potential specificity for tumor cells, indicating that it may not enhance the clinical outcome associated with ATO monotherapy in vivo.     

Newin vitro fluorimetric microtitration assays for toxicological screening of drugs

Flow cytometry has been widely used to quantify fluorescent probes in cell culture. However, FCM is not adapted to toxicological screenings due to the cost, the length and the poor reproducibility of this technique. Moreover, several multicenter studies have preferred microtitration methodologies for drug screening. A new fluorimetric technology has been designed that is sensitive and adapted to direct screening in 96-well microplates. This fluorimeter uses cold light technology (CLF) with chemical and physical modifications of the lighting system (Rat et al., 1995). CLF allows reading of UV, visible and near infrared fluorescence by increasing light energy (from 1000 to 2300 lumens) and reducing the calorific part of light (IR>900 nm, Joule effect). It induces a decrease in background and a 500- to 1000-fold improvement of detection limit of probes in comparison with classical fluorimeters and permits detection of pg/ml to fg/ml. CLF allows easy evaluation of cell injury induced by physical agents (UVA) or chemical toxins (CCl₄). Four biological endpoints for cytotoxicity evaluation have been tested with several probes: proliferation (H33258); viability (fluorescent Neutral Red); cell-cell adhesion (calcein-AM); and mitochondrial metabolic effects (Rhodamine 123). Rh 123 assay appeared more sensitive than fluorimetric or photometric detection of Neutral Red assay. Cold light fluorimetry (CLF) permits direct detection of low concentrations of probes (pg/ml to fg/ml). CLF is shown to improve classical cytotoxicity assays and, owing to its adaptability to microtitration (in 6-, 12- or 96-well plates and in Petri dishes), it is thus a promising alternative to flow cytometry for drug cytotoxicity screening.Abbreviations CLF cold light fluorimetry - FCM flow cytometry - H33258 Hoechst 33258 - IR infrared - NIR near infrared - UV ultraviolet - MTT tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide) - Rh123 Rhodamine 123     

In vitro biocompatibility of denture relining materials

Objective: The aim of the present study was to evaluate the in vitro biocompatibility of denture relining materials using cell culture tests and a test for irritation mechanisms. Background: Denture relining materials contain non‐reacted constituents that may leach out during use inducing local toxic or irritative effects. Materials and methods: One chemically cured, four visible light cured and five dual‐cured products were included. Cured test specimens were used for the filter diffusion test, and extracts of cured specimens were applied in the MTT and the irritation test using the hen's egg test‐chorioallantoic membrane (HET‐CAM) method. Results: Five of the tested materials were slightly or moderately cytotoxic in the filter diffusion test, and one product coated with a liner induced severe toxicity. Cell cultures incubated for 24 hour with the test samples were more damaged than those incubated for 2 hour. In the MTT test, extracts of nine of the 11 products induced cytotoxicity. No extracts showed irritation, whereas the coating and two bonding agents tested were strong irritants. Conclusion: Most of the tested materials contained water soluble, toxic substances that leach out of the products and that some time was needed to obtain cytotoxic amounts of the leachables. Many dental materials elicit cytotoxic response, but this does not necessarily reflect the long‐term risk for adverse effects as the oral mucosa is generally more resistant to toxic substances than a cell culture.     

Antioxidant activity of benzoxazolinonic and benzothiazolinonic derivatives in the LDL oxidation model

A series of benzazolone compounds were synthesized utilizing benzoxazolinonic and benzothiazolinonic heterocycles as the building unit. The antioxidant activity of these compounds was determined by inhibition of lipid peroxidation. The oxidation of LDL was induced in the presence of CuSO₄ or 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The protective action of these compounds against the cytotoxicity was evaluated with lactate dehydrogenase (LDH) activity in bovine aortic endothelial cells (BAECs) and cellular vitality by measuring mitochondrial activity in the presence of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The best antioxidant activities were observed for phenolic compounds 13 and 14b with ratio R = 2.5, 3.2 (5 μM). Both of these test substances increased the cell viability significantly as indicated by MTT assay and LDH release assay.     

Cross contamination associated with the use of multiwell culture plates for cytotoxicity assessment of volatile chemicals

In vitro toxicity testing can involve technical problems due to the evaporation of volatile test chemicals. The cytotoxicity of two volatile chemicals (butanol and ethanol has been assessed with neutral red assay in conventional microtiter plates. The non volatile DMSO chemical is used as a negative control. Under these conditions, an important cross contamination between test concentration groups has been observed. This affects cytotoxicity estimation which is overestimated. This cross contamination is prevented when special plates containing removalbe bars are used.     

Mutation detection in the breast cancer gene BRCA1 using the protein truncation test

About 400 distinct mutations have been defined in the BRCA1 gene, and these are spread fairly evenly through the 5592 bp of coding DNA. This circumstance presents a formidable challenge for mutation screening. Apart from total direct sequencing, the preferred screening method has been single-strand conformation polymorphism (SSCP) gels, with a smaller input from constant denaturant gradient electrophoresis (CDGE), heteroduplex (HD) analysis, and mismatch cleavage. The protein truncation test (PTT) was used early in BRCA1 mutation screening but has not been widely adopted, perhaps because a straightforward analysis of the whole BRCA1 gene requires working with RNA and all its perceived problems. The present work was undertaken to assess the practicality of using the PTT under routine conditions for the screening of long genes such as BRCA1 that are not highly expressed in lymphocytes. We conclude that, provided RNA preparation is carried out effectively and consistently, the PTT approach has significant advantages over other methodologies such as SSCP gels.     

Detection of 3-nitropropionic acid and cytotoxicity inMucor circinelloides

Mucorales are regarded as the aetiological agents of Mucormycosis. Their capabilities to produce mycotoxins are not profoundly investigated, in contrast to those of the fungi from the generaPenicillium, Aspergillus, orFusarium. The aim of this study was to isolate and identify fungi of the order Mucorales and investigate mycotoxins production. Twelve samples of visibly moulded grass silage and eight samples of damaged whole crop maize silage were analysed. Malt extract agar plates were used for sub cultivation. Three fungal species of the order Mucorales were isolated from grass silage, which were identified by their macro-and micro-morphology asAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer. The cytotoxicity ofMucor circinelloides extract was analysed using the cytotoxicity test (MTT assay) and the result, showed a low cytotoxicity. Additionally extracts fromAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer were tested for mycotoxin-production using an LC/MS/MS-based multimycotoxin method. 3-nitropropionic acid was detected in the culture extract ofMucor circinelloides. Presented at the 30^th Mykotoxin Workshop Utrecht, Netherlands, April 28–30, 2008.     

A critical comparison of three internalization assays applied to the evaluation of a given mAb as a toxin-carrier candidate

In the attempt to define a strategy for screening new monoclonal antibodies (mAb) that could be appropriate for clinical application in oncology, we evaluated the suitability of three methods: a direct internalization assay (DIA), an indirect internalization assay (IIA) and an indirect cytotoxicity assay (ICA), by applying them to already selected mAb. The latter were directed against three antigenic systems [38-kDa glycoprotein (gp38), epidermal growth factor receptor, and theneu oncogene product], which, according to their tumor selectivity, could be considered suitable for mAb-guided therapy. The dose-dependent and time-dependent binding, as well as the low intraassay variability, demonstrated the reliability of the three tests. However, a certain degree of inter-assay variability was observed in each one, the highest value being that found when IIA was applied. Furthermore, the degree of variability, as well as the predictability, seemed to be more related to the mAb/antigen (Ag) combination used rather than to the test applied. From the overall data we suggest a procedure to be applied for screening purposes. As a first approach applied to the raw material, ICA is only suitable for screening in the case of an already selected toxin whereas IIA may be helpful to eliminate the true negative mAb. After purification of the relevant mAb a repeated analysis using DIA could allow the selection of true internalizing mAb. However, this second screening should be followed by a further analysis of the fate of the Ag–Ab complex after internalization.     

Evaluation of the cytotoxicity of a two photon absorbing fluorescence compound on human HepG2 cells and its application to tracking human hepatic cancer cells in mice

Abstract

Small organic dyes have been applied widely in fluorescence imaging techniques for biomedical research. We investigated the cytotoxicity of a novel fluorescent dye, trans-4-(N-2-hydroxyethyl-N-ethyl amino)-4′-(dimethyl amino) stilbene (DMAHAS), on human hepatocellular carcinoma (HepG2) cells using methyl thiazolyl tetrazolium(MTT), a neutral red assay, a Coomassie brilliant blue assay, and flow cytometric analysis. Our results showed that DMAHAS had live cell permeability, stable cytosolic localization and no significant cytotoxicity to HepG2 cells. We explored its application further for tumor cell tracking in a human liver tumor xenograft mouse model. Tumor xenografts were examined by fluorescence imaging and conventional histological methods. In addition, a method based on DMAHAS release was developed for tumor-specific cytotoxicity analysis. Our study indicated that DMAHAS is a reliable probe for tumor tracking and fluorescence imaging.     

Synthesis and cytotoxicity evaluation of 22,23-oxygenated stigmastane derivatives

Starting from (22E)-3alpha,5alpha-cyclo-6beta-methoxystigmast-22-ene eighteen derivatives of (22S,23S)-22,23-oxidostigmastane, (22R,23R)-22,23-oxidostigmastane, and (22R,23R)-22,23-dihydroxystigmastane were synthesized and screened for cytotoxicity in human hepatoma Hep G2 cells and human breast carcinoma MCF-7 cells using MTT assay. Four compounds of this series exhibited high cytotoxicity in both cells; three compounds were selectively toxic in MCF-7 cells, one compound was toxic in Hep G2 cells, rather than in MCF-7 cells; four compounds at low concentrations increased MTT test values over the control.     

Synthesis,characterization, and anticancer evaluation of novel 4-hydrazinothiazole analogs

Single-step synthesis of novel 4-hydrazinothiazole derivatives 6a–e was achieved under mild conditions using the sequential four-components method involving isothiocyanate, aminoguanidine, carbonyl adduct, and α-haloketone derivatives. Deprotection of these hydrazinothiazoles was influenced by acylation, providing a novel group of diacylated molecular structures with a broader scope for the design of thiazolyl-containing drugs 7a and 7b . FTIR, ¹H/¹³C NMR, LC–MS spectroscopy, and CHN elemental analyses were used to study the compound chemical structures. Using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human periodontal ligament fibroblast (HPDLF) cells, the 4-hydrazinothiazole derivatives were screened for cytotoxicity in an in vitro cytotoxicity investigation. The 4-hydrazinothiazole compound 6b bearing an isopropylidene-hydrazino group demonstrated strongly potent cytotoxicity against CAKI1 (IC₅₀ = 1.65 ± 0.24 μM) and A498 (IC₅₀ of 0.85 ± 0.24 μM). Furthermore, the chloroacetyl-containing thiazole compound 7a displayed efficient inhibition of growth against the test cell lines CAKI1 and A498 at low micromolar concentrations, IC₅₀ 0.78 and 0.74 μM, respectively.     

Cytotoxicity of extracts from Dolsan leaf mustard Kimchi treated with lactic acid bacteria on lung and gastric cancer cells

The cytotoxicity of extracts from Dolsan leaf mustard Kimchi (DLMK) treated with lactic acid bacteria on A 549 human lung cancer cells and SNU-601 human gastric cancer cells were investigated. Leuconostoc mesenteroides, Leu. Gelidum, and Weissella kimchii previously isolated from properly ripened DLMK were inoculated to DLMK as a starter (1 × 10⁸ CFU/mL). The DLMK was then fractionated by various extracting solvents. The cytotoxicity of MeOH extracts from DLMK on A 549 and SNU-601 cancer cells was found to occur in a dose-dependent manner. Although the cytotoxicity of the MeOH extracts was found to be approximately 20 to 30% at concentrations of 250 μg/mL by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide) assay, cytotoxicity of chloroform soluble fraction of DLMK treated with W. kimchii showed about 80 to 90%. Consequently, the growth of cancer cells was inhibited significantly in medium containing DLMK extracts. In addition, significant morphological changes such as cell condensation, cell fragmentation, and alterations in the size and shape of the cells were observed in cells grown in medium that contained the DLMK extracts. Taken together, these results suggest that inhibition of the proliferation of cancer cells by apoptosis was induced by DLMK extracts.