The Use of Human Lymphocytes for the In Vitro Estimation of Acute Toxicity of Ten Chemicals from the MEIC List

1991 ◽  
Vol 19 (2) ◽  
pp. 187-190
Author(s):  
Mette Tingleff Skaanild ◽  
Jørgen Clausen
Keyword(s):  
Hela Cells ◽  
Cultured Cells ◽  
Human Lymphocytes ◽  
Primary Cultures ◽  
Biological Significance ◽  
In Vitro Cytotoxicity ◽  
In Vitro Toxicity ◽  
Human Hepatoma Cells ◽  
Response Curves

The acute cytotoxicity of ten chemicals included in the MEIC (multicentre evaluation of in vitro cytotoxicity) list has already been estimated in various cell lines, e.g. primary rat hepatocytes, HeLa cells, human hepatoma cells, and 3T3 and mouse fibroma cells. In the present study, primary cultures of human lymphocytes, with or without an S9-mix (microsomes), were used to assay for in vitro toxicity. The cultured cells were initially treated with different concentrations of the respective drugs for 24 hours. Then cellular enzymatic activity was estimated using two assay systems, namely, measurement of cytosolic LDH activity and the mitochondrial (diaphorase) MTT test. Since the biological significance of the assay of LC50 values is dependent upon the slope of the dose-response curves, the results are expressed as LC20, LC50 and LC80 values in μM for the ten drugs. A Spearman rank correlation analysis revealed a significant correlation between the results from the two assays in both cell culture systems (r=1.00 and r=0.99). The LC50 values found in the lymphocyte cultures, both with and without an S9-mix, correlated well with the results previously found using primary cultures of hepatocytes and HeLa cells.

Protein Precipitation In Vitro as a Measure of Chemical-induced Cytotoxicity: An EDIT Sub-programme

2001 ◽  
Vol 29 (3) ◽  
pp. 309-324
Author(s):  
Apolonia Novillo ◽  
Barbro Ekwall ◽  
Argelia Castaño
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Exposure Period ◽  
Negative Control ◽  
In Vitro Cytotoxicity ◽  
Protein Precipitation ◽  
In Vitro Toxicity ◽  
Human Cell Lines ◽  
Response Curves

As a priority area of the Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT) programme, an in vitro protein precipitation (PP) assay was used on the 50 reference chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to confirm and extend the MEIC results. Dose–response curves were generated for only 30 of the chemicals, and the concentrations causing 10% (EC10) and 50% (EC50) protein precipitation versus the positive control were chosen as endpoints. The number of chemicals with a positive response increased to 46 when a new endpoint, the minimum effect concentration (MEC) that induces protein precipitation with respect to the negative control, was used. When the results were correlated with in vitro cytotoxicity in human cell lines, a similarly good correlation was found between the various endpoints of the PP assay at 5 hours and the 24-hour IC50 average cytotoxicity in human cell lines, even though the number of chemicals included in the correlation was larger for the MEC. Using the prediction error, the endpoint that gave the best correlation between the PP assay and human cell cytotoxicity was once more found to be the 5-hour MEC, and this was chosen for the PP assay. The sensitivity of the PP assay is lower than that of the in vitro cell-line cytotoxicity assay, possibly due to its shorter exposure period and because precipitation is the ultimate event in the sequence of a protein disturbance. It is expected that earlier denaturation steps would give better sensitivity. However, this simple, inexpensive and rapid assay could be useful in the early stages of testing chemicals.

In Vitro Cytotoxicity Testing: 24-hour and 72-hour Studies with Cultured Lung Cells

1993 ◽  
Vol 21 (2) ◽  
pp. 167-172
Author(s):  
Desirée Hopkinson ◽  
Rae Bourne ◽  
Frank A. Barile
Keyword(s):  
Cultured Cells ◽  
Linear Regression Analysis ◽  
Biological Significance ◽  
Culture Method ◽  
In Vitro Cytotoxicity ◽  
Lung Epithelial Cells ◽  
Blood Concentrations ◽  
Lung Epithelial ◽  
Ic50 Values

This study was designed to evaluate the potential of an in vitro cell culture method for its ability to determine cytotoxicity and to compare the cytotoxic concentrations with established LD50 values for the same chemicals. Rat lung epithelial cells (L2) were incubated in the absence or presence of increasing concentrations of the test chemical for 24 hours, and the inhibition of incorporation of radio-labelled amino acids into newly synthesised proteins was used as a marker for toxicity. In addition, cultured cells were exposed to the test chemicals for 72 hours, and cell proliferation experiments were performed as parallel measures of toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. The biological significance of the results of testing 20 chemicals shows that the experimental IC50 values are as accurate as predictors of human toxicity as are equivalent toxic blood concentrations derived from rodent LD50s. Results obtained from 72-hour growth studies reveal a greater sensitivity to cytotoxicity than from the 24-hour protein synthesis experiments. Statistically, however, the differences between the two protocols are inconclusive. It is anticipated that these procedures, together with a related battery of tests, may supplement or replace animal protocols currently used for human risk assessment.

MEIC Evaluation of Acute Systemic Toxicity

1996 ◽  
Vol 24 (1_part_1) ◽  
pp. 251-272 ◽  
Author(s):  
Cecilia Clemedson ◽  
Elisabeth McFarlane-Abdulla ◽  
Marianne Andersson ◽  
Frank A. Barile ◽  
Mabel C. Calleja ◽  
...  
Keyword(s):  
Full Range ◽  
Primary Cultures ◽  
In Vitro Cytotoxicity ◽  
Systemic Toxicity ◽  
Toxicity Tests ◽  
In Vitro Toxicity ◽  
Blood Concentrations ◽  
Differentiated Cells ◽  
Basal Cytotoxicity

The multicentre evaluation of in vitro cytotoxicity (MEIC) study is a programme designed to evaluate the relevance of in vitro toxicity tests for predicting human toxicity, and is organised by the Scandinavian Society for Cell Toxicology. The project started in 1989 and is scheduled to be finished by June 1996. MEIC is a voluntary effort by international laboratories to test the same 50 reference chemicals in their own in vitro toxicity systems. At present, 31 laboratories have submitted results for the first 30 reference chemicals from a total of 68 in vitro cytotoxicity tests. In the definitive evaluation of the MEIC programme, these in vitro results will be compared with human lethal blood concentrations and other relevant acute systemic toxicity data, and the results will be published as a series of articles. This paper, which is the first article in this series, describes and analyses the methodologies used in the 68 tests. The origins and purities of the test chemicals, the biological systems and the toxicity endpoints are also discussed. Since MEIC is not centrally directed, the selection of tests was entirely dependent on the preferences of the individual laboratories. Thus, the collection of tests is not representative of the full range of existing in vitro toxicity tests. In our study, basal cytotoxicity tests and ecotoxicological tests are prevalent, while tests for toxicity to primary cultures of differentiated cells, measured by organotypic toxicity endpoints, are clearly under-represented.

scholarly journals The in vitro assessment of the toxicity of volatile, oxidisable, redox-cycling compounds: phenols as an example

2021 ◽  
Author(s):  
Laia Tolosa ◽  
Teresa Martínez-Sena ◽  
Johannes P. Schimming ◽  
Erika Moro ◽  
Sylvia E. Escher ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Degradation Products ◽  
Degradation Kinetics ◽  
Redox Cycling ◽  
In Vitro Toxicity ◽  
Cross Contamination ◽  
Physicochemical Changes ◽  
In Vitro Assessment ◽  
Biological Performance

AbstractPhenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it. TMHQ has fast degradation kinetics followed by significant diffusion rates of the resulting quinone to adjacent wells, other degradation products being able to air-diffuse as well. DTBEP showed lower degradation kinetics, but a higher diffusion rate. In both cases the in vitro toxicity was underestimated because of a decrease in concentration, in addition to cross-contamination to neighbouring wells. We identified four degradation products for TMHQ and five for DTBEP indicating that the current effects measured on cells are not only attributable to the parent phenolic compound. To overcome these drawbacks, we investigated in detail the physicochemical changes occurring in the course of the incubation and made use of gas-permeable and non-permeable plastic seals to prevent it. Diffusion was greatly prevented by the use of both plastic seals, as revealed by GC–MS analysis. Gas non-permeable plastic seals, reduced to a minimum compounds diffusion as well oxidation and did not affect the biological performance of cultured cells. Hence, no toxicological cross-contamination was observed in neighbouring wells, thus allowing a more reliable in vitro assessment of phenol-induced toxicity.

Neurotoxicity of Seven MEIC Chemicals Evaluated in Organotypic Cultures of Chick Embryonic Dorsal Root Ganglia

1997 ◽  
Vol 25 (3) ◽  
pp. 303-309
Author(s):  
Václav Mandys ◽  
Katerina Jirsová ◽  
Jirí Vrana
Keyword(s):  
Toxic Effect ◽  
Neurite Outgrowth ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Growth Parameters ◽  
In Vitro Cytotoxicity ◽  
Organotypic Cultures ◽  
Response Curves ◽  
Agar Culture

The neurotoxic effects of seven selected Multicenter Evaluation of In Vitro Cytotoxicity programme chemicals (methanol, ethanol, isopropanol, sodium chloride, potassium chloride, iron [II] sulphate and chloroform) were evaluated in organotypic cultures of chick embryonic dorsal root ganglia (DRG), maintained in a soft agar culture medium. Two growth parameters of neurite outgrowth from the ganglia — the mean radial length of neurites and the area of neurite outgrowth — were used to evaluate the toxicities of the chemicals. Dose-dependent decreases of both parameters were observed in all experiments. IC50 values (the concentration causing 50% inhibition of growth) were calculated from the dose-response curves established at three time-points during culture, i.e. 24, 48 and 72 hours. The lowest toxic effect was observed in cultures exposed to methanol (the IC50 ranging from 580mM to 1020mM). The highest toxic effect was observed in cultures exposed to iron (II) sulphate (the IC50 ranging from 1.2mM to 1.7mM). The results of other recent experiments suggest that organotypic cultures of DRG can be used during in vitro studies on target organ toxicity within the peripheral nervous system. Moreover, these cultures preserve the internal organisation of the tissue, maintain intercellular contacts, and thus reflect the in vitro situation, more precisely than other cell cultures.

scholarly journals Synthesis of Gold Nanoparticles by Using Green Machinery: Characterization and In Vitro Toxicity

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 808
Author(s):  
Ahmed Al Saqr ◽  
El-Sayed Khafagy ◽  
Ahmed Alalaiwe ◽  
Mohammed F. Aldawsari ◽  
Saad M. Alshahrani ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Average Particle Size ◽  
In Vitro Cytotoxicity ◽  
In Vitro Toxicity ◽  
Cervical Cancer Cell Line ◽  
Average Particle ◽  
Anticancer Activities ◽  
Tem Analysis ◽  
Benincasa Hispida

Green synthesis of gold nanoparticles (GNPs) with plant extracts has gained considerable interest in the field of biomedicine. Recently, the bioreduction nature of herbal extracts has helped to synthesize spherical GNPs of different potential from gold salt. In this study, a fast ecofriendly method was adopted for the synthesis of GNPs using fresh peel (aqueous) extracts of Benincasa hispida, which acted as reducing and stabilizing agents. The biosynthesized GNPs were characterized by UV–VIS and Fourier transform infrared spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering. In addition, the in vitro antibacterial and anticancer activities of synthesized GNPs were investigated. The formation of gold nanoparticles was confirmed by the existence of a sharp absorption peak at 520 nm, corresponding to the surface plasmon resonance (SPR) band of the GNPs. TEM analysis revealed that the prepared GNPs were spherical in shape and had an average particle size of 22.18 ± 2 nm. Most importantly, the synthesized GNPs exhibited considerable antibacterial activity against different Gram-positive and Gram-negative bacteria. Furthermore, the biosynthesized GNPs exerted remarkable in vitro cytotoxicity against human cervical cancer cell line, while sparing normal human primary osteoblast cells. Such cytotoxic effect was attributed to the increased production of reactive oxygen species (ROS) that contributed to the damage of HeLa cells. Collectively, peel extracts of B. hispida can be efficiently used for the synthesis of GNPs, which can be adopted as a natural source of antimicrobial and anticancer agent.

Differential in vitro effects of chemotherapeutic agents on primary cultures of human ovarian carcinoma

2004 ◽  
Vol 14 (4) ◽  
pp. 607-615 ◽  
Author(s):  
P. Kornblith ◽  
R. L. Ochs ◽  
A. Wells ◽  
M. J. Gabrin ◽  
J. Piwowar ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Human Tumor ◽  
Clinical Results ◽  
Chemotherapeutic Agents ◽  
Primary Ovarian Cancer ◽  
In Vitro Effects ◽  
Therapeutic Decision Making

The treatment of ovarian cancer principally relies on the use of platinum and taxane chemotherapeutic agents. Short-term clinical results have been encouraging, but long-term responses remain limited. In this report, an in vitro assay system that utilizes cells grown from human tumor explants has been used to quantitatively evaluate responses to relevant concentrations of alternative chemotherapeutic agents. The results suggest that there are significant differences in the responses of explant-derived cultured cells to the different agents tested. In an evaluation of 276 primary ovarian cancer specimens, five nonstandard drugs were tested in 51 cases. Of these 51 cases, cyclophosphamide had the highest rate of response at 67%, followed by doxorubicin at 61%, gemcitabine at 49%, etoposide at 48%, and topotecan at 14%. Venn diagrams, representing the in vitro responses to the platins and taxanes, as well as the responses to the nonstandard drugs, illustrate that there clearly are distinct differences among patients in a given population. These data underscore the potential importance of evaluating each patient's response to a number of different drugs to optimize the therapeutic decision-making process.

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