Neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells to predict human toxicity

2002 ◽  
Vol 22 (1) ◽  
pp. 61-65 ◽  
Author(s):  
Paul J. Dierickx ◽  
Ellen M. Scheers
Keyword(s):  
Neutral Red ◽  
Human Toxicity ◽  
Uptake Inhibition ◽  
Neutral Red Uptake ◽  
Rat Hepatoma

Cytotoxicity of Amino Alcohols to Rat Hepatoma-derived Fa32 Cells

2002 ◽  
Vol 30 (3) ◽  
pp. 309-312
Author(s):  
Ellen M. Scheers ◽  
Anna Forsby ◽  
Paul J. Dierickx
Keyword(s):  
Vitamin E ◽  
Phenyl Group ◽  
Amino Alcohols ◽  
Neutral Red ◽  
Glutathione Depletion ◽  
Hydroxyl Group ◽  
Racemic Mixture ◽  
Uptake Inhibition ◽  
Neutral Red Uptake ◽  
Rat Hepatoma

Amino alcohols are used as emulsifying agents in dry-cleaning soaps, wax removers, cosmetics, paints and insecticides. The cytotoxicities of 12 amino alcohols, which differed in chain length, position of the amino and alcohol groups, and the presence of an additional phenyl group, were determined by the neutral red uptake inhibition assay with normally cultured, glutathione-depleted or antioxidant-enriched Fa32 rat hepatoma-derived cells. Glutathione depletion and antioxidant enrichment were achieved by including 50μM L-buthionine- S,R-sulphoximine (BSO) or 100μM α-tocopherol acetate (vitamin E) in the culture medium for 24 hours before and during the assay. The cytotoxicity of the amino alcohols observed after treatment for 24 hours was expressed as the concentration of compound needed to induce a 50% reduction in neutral red uptake (NI50). The observed NI50 values ranged from 3mM to 30mM. The individual stereoisomers and a racemic mixture of 1-amino-2-propanol exhibited similar cytotoxicities (with normally cultured Fa32 cells, and vitamin E- and BSO-treated cultures). Similar NI50 values for D-(+)-2-amino-1-propanol, 3-amino-1-propanol and the L-, D- or DL- forms of 1-amino-2-propanol, indicated that the position of the amino group had little influence on the cytotoxicities of the amino alcohols. In contrast, the position of the hydroxyl group appeared to play an important role for the toxicity of the compound, as indicated by the significantly different NI50 values for 4-amino-1-butanol and 4-amino-2-butanol. An additional phenyl group greatly increased the cytotoxicity of 2-amino-1,3-propanediol. For most of the compounds, cytotoxicity increased when GSH was depleted, and decreased when the cells were enriched with vitamin E. This indicated that most of the tested chemicals interact with GSH, either directly or indirectly, by processes which generate oxygen free-radicals. Decreased toxicity was found for most of the chemicals administered to vitamin E-enriched cells, indicating that reactive oxygen species could be involved in the toxicity of the amino alcohols.

Glutathione protection against hydrogen peroxide, tert-butyl hydroperoxide and diamide cytotoxicity in rat hepatoma-derived Fa32 cells

1999 ◽  
Vol 18 (10) ◽  
pp. 627-633 ◽  
Author(s):  
P J Dierickx ◽  
G Van Nuffel ◽  
I Alvarez
Keyword(s):  
Neutral Red ◽  
Therapeutic Effects ◽  
Ozone Therapy ◽  
Uptake Inhibition ◽  
Butyl Hydroperoxide ◽  
Tert Butyl ◽  
Time Period ◽  
Tert Butyl Hydroperoxide ◽  
Neutral Red Uptake ◽  
Rat Hepatoma

1 Several ozonides, peroxides and aldehydes are formed during ozone therapy, recently introduced in medicine. tert-Butyl hydroperoxide (t-BHP), H2O2 and diamide were investigated as model substrate in rat hepatoma-derived Fa32 cells. 2 The cytotoxicity was measured by the neutral red uptake inhibition assay after 1 h or 24 h treatment. The relative toxicities were quantified by the determination of the NI50. This is the concentration of test compound required to induce an inhibition of 50% in neutral red uptake as compared to the control cells. All test chemicals were more toxic after 24 h than after 1h. 3 The influence of the glutathione (GSH) alteration on the cytotoxicity was measured by treating the cells with 2-oxo-4-thiazolidine carboxylic acid (OTC) or L-buthio-nine sulfoximine (BSO). OTC increased the endogenous GSH content in the cells. BSO pretreatment strongly decreased the NI50 of the three chemicals. OTC pretreatment increased the NI50 of H2O2 but not of t-BHP and diamide. This can be explained by the strong GSH-depletion after 1 h by t-BHP and diamide, which contrasted with a weak GSH-depletion by H2O2 after the same time period. 4 The three test chemicals increased the endogenousGSH content after 24 h. t-BHP and H2O2,butnot diamide, increased the total GSH transferase (GST) activity. Several alterations of the GST subunits were observed. Most striking was the increase of class alpha GST subunits, also for diamide. 5 SinceH2O2 and t-BHP are ozone metabolites thought to be responsible for the therapeutic effects of well-dosed ozone, the results show that Fa32 cells can be used as a valuable alternative model system for studying the effects encountered in human ozone therapy.

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.

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.

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.

scholarly journals Cytotoxicity of Some Nitroimidazole Derivatives - Comparative Studies on Human and Rat Hepatoma Cell Lines

2012 ◽  
Vol 56 (4) ◽  
pp. 579-584 ◽  
Author(s):  
Lidia Radko ◽  
Maria Minta
Keyword(s):  
Cell Lines ◽  
Hepatoma Cell ◽  
Neutral Red ◽  
Total Protein Content ◽  
Cytotoxic Potential ◽  
Hepatoma Cell Lines ◽  
Mtt Reduction ◽  
Neutral Red Uptake ◽  
Toxic Concentrations ◽  
Rat Hepatoma

Abstract The cytototoxic potential of metronidazole, tinidazole, ronidazole, and ornidazole, using human and rat hepatoma cell lines (HepG2 and FaO) in culture was assessed. The cells were treated with drugs for 24, 48 and 72 h at 37 °C in 5% CO2 at concentrations of 0.1 to 200 μg/mL. Following the treatment period, the cells were assayed by four independent assays: MTT reduction, neutral red uptake (NRU), total protein content (TPC), and LDH leakage. The results suggest that nitroimidazoles are of low cytotoxic potential (EC50 >200μg/mL). The exception was ronidazole, which demonstrated a distinct endpoint sensitivity related to the species. EC50 (μg/mL) in human cells were: in MTT assay - 196±5.5 and 122±9.3 at 24 and 48 h, respectively, and in NRU assay - 150±1.25 at 72 h. Based on minimal toxic concentrations (EC20) for ronidazole, determined by all methods used in HepG2 cells, it could be concluded that their sensitivity was as follows: MTT>NRU>LDH>TPC.

scholarly journals Biological Innocuousness of a Device developed to select the Optimum Spermatozoa to be used in the Treatment of Infertility

2021 ◽  
Author(s):  
Marisa A Cubilla ◽  
Hector A Guidobaldi ◽  
Laura C Giojalas
Keyword(s):  
Colony Formation ◽  
Physiological State ◽  
Neutral Red ◽  
Sperm Selection ◽  
Neutral Red Uptake

The sperm selection assay (SSA) is a method based on chemotaxis to obtain spermatozoa at the optimum physiological state to successfully fertilize the egg. It consists of a device made of acrylic and an attractant solution which includes progesterone. We evaluate potential cytotoxicity interactions by means of Neutral Red uptake, the MTT and colony formation assays, according to ISO normative. Here we showed that even stressing the conditions of the assays, the SSA device alone or together with the progesterone solutions employed, showed to be innocuous for the cells. Suggesting that SSA could be incorporated into the ART procedures.

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