The EYTEX™ System and the Neutral Red Uptake Inhibition Assay in Cultured Hep G2 Cells as Alternative Methods for In Vivo Eye Irritation Following the EEC Protocol

1990 ◽  
Vol 17 (4) ◽  
pp. 325-333
Author(s):  
Paul J. Dierickx ◽  
Virginia C. Gordon
Keyword(s):  
Neutral Red ◽  
Alternative Methods ◽  
Inhibition Assay ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Eye Irritation ◽  
Uptake Inhibition ◽  
In Vitro Methods ◽  
Neutral Red Uptake

The neutral red uptake inhibition assay and the EYTEX™ system were investigated as alternative methods for the assessment of eye irritation, determined according to the EEC protocol. The 17 test chemicals used were mainly organic solvents. The xenobiotics were applied to Hep G2 cells for 24 hours at different concentrations. Neutral red uptake inhibition was then measured. The results are expressed as the NI50 value, which is the concentration of test compound required to induce a 50% reduction in neutral red uptake. The same chemicals were also tested as coded samples by the EYTEX™ test according to the manufacturer's directions. A nearly identical quantitative correlation was found for both in vitro methods with corneal opacity scores: r = 0.84 for EYTEX™ scores and r = 0.83 for log NI50, expressed in μg/ml. Whilst these correlations are certainly not perfect, it is clear that both in vitro methods can be used as valuable prescreening methods.

Evaluation of the In Vitro Uridine Uptake Inhibition Assay in Comparison with the In Vivo Eye Irritation Test as Prescribed by the EEC

1988 ◽  
Vol 15 (4) ◽  
pp. 290-296
Author(s):  
Guido A. Jacobs ◽  
Paul J. Dierickx ◽  
Mark A. Martens
Keyword(s):  
Screening Method ◽  
Corneal Opacity ◽  
Inhibition Assay ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Eye Irritation ◽  
Uptake Inhibition ◽  
Uridine Uptake ◽  
G2 Cells

In vivo eye irritation tests were carried out on 17 substances (mainly solvents) according to the experimental protocols laid down in the European Community legislation on dangerous substances and the OECD Guidelines. Erythema, chemosis, corneal opacity and iritis were observed and evaluated according to the interpretation rules laid down in EEC legislation. The uridine uptake inhibition assay was carried out on Human Hep G2 cells according to the method of Shopsis & Sathe (1), modified by Dierickx & Martens (2). Inhibition of uridine uptake was expressed as the UI50 (Hep G2 cells, mM). Comparison of the results of our two studies revealed only a poor relationship between the UI50 values and the mean scores obtained for corneal opacity (Spearman rank correlation; r=0.57, p<0.02). Consideration of just the monoalcohols, together with the ketones and the formamides, gave a good linear correlation between the UI50 (Hep G2 cells) and the <<Se>> (r=0.93). If a UI50 value of 50mM is accepted as a limit for classification of irritancy for the eye, no false positives or false negatives are obtained. The UI50 can only be used within strictly defined chemical classes, as a first screening method for detecting possible irritants.

Glutamic Acid Uptake Inhibition Assay in Cultured Hep G2 Cells as an Alternative Method for Evaluating Potential In Vivo Eye Irritation

1989 ◽  
Vol 16 (4) ◽  
pp. 344-352
Author(s):  
Paul J. Dierickx
Keyword(s):  
Glutamic Acid ◽  
Corneal Opacity ◽  
Acid Uptake ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Eye Irritation ◽  
Uptake Inhibition ◽  
Range Finding ◽  
G2 Cells

Glutamic acid (GA) content was measured in cultured Hep G2 cells, after treatment of the cells with test compounds. The results with 37 chemicals were compared with their respective rabbit eye irritation data, of which 17 were determined according to the OECD test, and the other 20 in range-finding studies. The chemicals were mainly organic solvents (alcohols, esters, amines, acids and others). The xenobiotics were applied to the cells for 4 hours at 5 different concentrations. The cells were then incubated for 15 minutes with tritiated GA. GA uptake inhibition was measured by liquid scintillation counting, and the results were expressed as the GI50 value, which is the concentration of test compound required to induce a 50% reduction in GA uptake. A linear correlation coefficient r = 0.66 was found between the log GI50 and the mean corneal opacity scores. This value is comparable to that obtained in total protein and uridine uptake inhibition studies. However, r = 0.81 was found when the log GI50 was compared with range-finding scores, indicating that a closer relationship exists between cytotoxicity and the latter.

The Influence of Glutathione on the Cytotoxicity of Metals in Rat Hepatoma-derived Fa32 Cells

1996 ◽  
Vol 24 (3) ◽  
pp. 399-403
Author(s):  
Paul J. Dierickx
Keyword(s):  
Hepatoma Cell ◽  
Neutral Red ◽  
Hepatoma Cell Line ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Glutathione S Transferase ◽  
Uptake Inhibition ◽  
Treatment Regimens ◽  
Rat Hepatoma ◽  
Good Agreement

The cytotoxicities of mercury, cadmium, nickel, cobalt, zinc and copper were investigated in rat hepatoma-derived Fa32 cells by using the neutral red uptake inhibition assay with three treatment regimens (2 hours, 24 hours and 1 week). Nickel and cobalt were almost non-toxic after 2 hours. Good correlations were observed between the 24-hour and the 1-week cytotoxicities, and cytotoxicity in human hepatoma-derived Hep G2 cells. L-buthionine-S,R-sulphoximine reduced the glutathione content to 5% after 24 hours. The cytotoxicity of the metals increased (3–12 times) in glutathione-depleted cells. A good agreement was demonstrated by HPLC between the glutathione S-transferase (GST) subunit composition in Fa32 cells and in rat liver, except that subunit 7 is also a major subunit in the hepatoma cell line. No evidence was obtained for an interaction of the GSTs in the glutathione-modulated cytotoxicity of the investigated metals.

A Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test

1998 ◽  
Vol 26 (5) ◽  
pp. 679-708 ◽  
Author(s):  
Horst Spielmann ◽  
Michael Balls ◽  
Jack Dupuis ◽  
Wolfgang J. W. Pape ◽  
Odile de Silva ◽  
...  
Keyword(s):  
Neutral Red ◽  
Optimum Concentration ◽  
Alternative Methods ◽  
Uv Filter ◽  
Eu Directive ◽  
Optimum Concentration Range ◽  
The Impact ◽  
Positive Results

In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1μg/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100μg/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1μg/ml and 10μg/ml, and that false positive results can be obtained at concentrations greater than 100μg/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100μg/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account.

The Hep G2 Cell Line as a Possible Alternative to Isolated Hepatocytes in Cytotoxicity Studies

1988 ◽  
Vol 16 (1) ◽  
pp. 16-22
Author(s):  
Marina Marinovich ◽  
Jose L. Lorenzo ◽  
Liliana M. Flaminio ◽  
Agnese Granata ◽  
Corrado L. Galli
Keyword(s):  
Cell Line ◽  
Rat Hepatocytes ◽  
Prostaglandin E ◽  
Human Hepatoma ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Isolated Rat Hepatocytes ◽  
G2 Cells ◽  
Isolated Rat

The hepatotoxicity of carbon tetrachloride (CC14) was evaluated in vitro in freshly isolated rat hepatocytes and in the human hepatoma cell line, Hep G2. Toxicity was assessed by the leakage of cytosolic enzymes (lactate dehydrogenase and aspartate aminotransferase) and cell viability (trypan blue exclusion). The established human cells were less sensitive to CCl4-induced injury; higher doses of the toxic agent and longer incubation times were necessary to elicit cell damage. Micromolar concentrations of prostaglandin E2 significantly decreased enzyme leakage in both Hep G2 cells and rat hepatocytes challenged with CC14; a stable derivative of prostacyclin (ZK 36374) was ineffective. These results suggest that human hepatoma Hep G2 cells may represent a valid alternative to isolated rat hepatocytes for an initial approach to the in vitro evaluation of cell toxicity.

Synthesis and Processing of Human Serum Apolipoprotein AII in vitro and in Hep G2 Cells

1985 ◽  
Vol 366 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Wilhelm STOFFEL ◽  
Rosemarie BLAU ◽  
Martin BURK
Keyword(s):  
Human Serum ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Synthesis And Processing ◽  
G2 Cells

The effect of hexaaza-and hexathia–macrocyclic ligands on transition metal cytotoxicity in human hepatoma-derived cultured cells

2002 ◽  
Vol 21 (8) ◽  
pp. 421-427 ◽  
Author(s):  
P W Smet ◽  
T F Pauwels ◽  
P J Dierickx
Keyword(s):  
Transition Metal ◽  
Cultured Cells ◽  
Copper Ions ◽  
Transition Metal Ions ◽  
Neutral Red ◽  
Macrocyclic Ligands ◽  
Human Hepatoma ◽  
Uptake Inhibition ◽  
Copper Zinc ◽  
Neutral Red Uptake

The effect of macrocyclic ligands on cytotoxic concentrations of the transition metal ions of copper, zinc, and cadmium was investigated. For this purpose, a hexaaza-[3,6,9,17,20,23-hexaazatricyclo[23.3.1.111,15] triaconta–1(29),11(30),12,14,25,27–hexaene (L2)] and hexathia-chelating ligand [1,4,7,10,13,16-hexathiacyclooctadecane (L3)] were used in the human hepatoma-derived HepG2 cell line. The cytotoxicity was measured by the neutral red uptake inhibition assay. First, the NI50 of the ligands, i.e., the concentration of the ligand inducing a 50% inhibition in neutral red uptake compared to control cells, was determined. In several metal/ligand combination experiments, the effects for L2 were difficult to interpret, whereas for L3 in combination with copper ions, a severe increase–and for zinc ions, a significant decrease of cell toxicity–relative to the metal control was observed. To further examine the different effects observed with L3 in combination with, respectively, Cu2+ and Zn2+, the glutathione (GSH) content was measured. The relative GSH content decreased as the concentration of L3 increased. It was proposed that the increased toxicity of the combination Cu2+ /L3 could be caused by the depletion of GSH and a subsequent inability to scavenge the produced reactive oxygen species (ROS). This hypothesis was supported by experiments during which vitamin E or C was added to the Cu2+ / L3 system.

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