The role of oxidative stress on the effect of 1,4,7,10,13,16-hexathiacyclooctadecane on copper and zinc toxicity in HepG2 cells.

Experiments have shown that 1,4,7,10,13,16-hexathiacyclooctadecane (L3) increased the Cu2+ toxicity on HepG2 cells, whereas the combination Zn(2+)/L3 was less toxic relative to the metal control. In all cases, glutathione (GSH) levels were decreased and vitamins C and E supplementation partially counteracted the increased toxicity in the Cu(2+)/L3-treated cells. The previously observed effects of this hexathiamacrocyclic ligand (L3) on the Cu2+ and Zn2+ toxicity were further investigated by first depleting the intracellular GSH levels by means of L-buthionine S,R-sulphoximine. Combined treatment with Cu(2+)/L3 resulted in complete cell death, whereas for Zn(2+)/L3 no severe effects were observed. Direct measurement of reactive oxygen species (ROS) revealed that Cu2+ induced a high degree of oxidative stress on the cells. This was not the case for Zn2+. The results proved a previously proposed mechanism in which GSH is used to conjugate the metal-ligand complex, but as a result of this, GSH is no longer available for inactivation of ROS. Also, both the intracellular copper and zinc content were determined for each experiment by means of inductively coupled plasma-atomic emission spectroscopy. According to these data, zinc is depleted in Cu(2+)/L3-treated cells, which could have consequences on superoxide dismutase and as a result of this on the amount of oxidative stress.

Formation of reactive oxygen species in rat hepatoma-derived Fa32 cells to predict human toxicity.

The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the increased reactive oxygen species (ROS) formation in rat hepatoma-derived Fa32 cells. ROS formation was measured with 2',7'-dichlorodihydrofluorescein diacetate as a fluorescent probe. The results were quantified by determining the ROS50. This is the concentration of test compound required to increase the ROS formation with 50% compared with control cells. An extremely high ROS formation was observed with ferrous sulfate. Of a total of 44 chemicals, an increased ROS formation was observed for 24. This was not the case for the 20 other chemicals. When the ROS formation in Fa32 cells was compared with human toxicity, the correlation coefficient was clearly higher than for human hepatoma-derived Hep G2 cells, at least when the extremely sensitive ferrous sulfate was withdrawn from the comparison. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Consequently, the ROS formation assay in Fa32 cells has a high predictive value for human toxicity, with the drawback that only ROS increasing chemicals can be evaluated.

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

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[23.3.1.1(11,15)] 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 Cu(2+)/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 Cu(2+)/L3 system.

Cytotoxicity of the MEIC reference chemicals in antioxidant-enriched, rat hepatoma-derived Fa32 cells.

Since vitamin E increases the antioxidant status of cells, its influence on cytotoxicity was investigated. The neutral red uptake (NRU) inhibition effects of 39 MEIC reference chemicals were measured after treatment of rat hepatoma-derived Fa32 cells in the presence of vitamin E for 30 minutes. The results were quantified in terms of the NI50, the concentration of test compound required to reduce the NRU by 50%. Sodium chloride was the only chemical that was more toxic in the presence of vitamin E. This effect was related to the concentration of vitamin E in the cell culture medium. A vitamin E dose-related response was also observed for the decreased toxicity of paracetamol and caffeine. Glutathione levels were slightly increased in the presence of vitamin E, which could contribute to the protective effect of vitamin E. Of the remaining chemicals, 50% were less toxic in the presence of vitamin E, but the correlation with the acute human toxicity data of the MEIC study was not improved. The results imply that reactive oxygen species interfere with the toxicity of a high proportion of toxic chemicals. The assay described provides a quick and easy method for checking whether reactive oxygen species contribute to the toxicity of a chemical.

Glutathione S-transferases of Aulacorthum solani and Acyrthosiphon pisum: partial purification and characterization.

Glutathione S-transferases (GST) play an important role in the detoxification of many substances including allelochemicals from plants. Brassicaceae plants contain glucosinolates and emit volatile isothiocyanates which affect the GST system. A comparison of the GST of two aphid species, the generalist Aulacorthum solani found on Brassicaceae and the Fabaceae specialist Acyrthosiphon pisum, was made to try to explain their respective feeding behaviour. Differences of GST were determined among the two aphid species based on purification by affinity chromatography, SDS-PAGE and on kinetic studies. Purification yields using an epoxy-activated Sepharose 6B column were highly different for the two aphid species (18% and 34% for A. solani and A. pisum, respectively). These variations were confirmed by SDS-PAGE. While only a 27-kDa band was observed for A. pisum, two bands of approximately 25-kDa were visualized for the generalist aphid, A. solani. Considering the kinetic results, differences of Km and Vmax were observed following the aphid species when a range of substrates (CDNB and DCNB) and GSH concentrations were tested. Studies on the detoxification enzymes of generalist and specialist herbivores would be undertaken to determine accurately the effect of the host plant on the organisms eating them, particularly in terms of biochemical and ecological advantages.

In vitro long-term cytotoxicity testing of 27 MEIC chemicals on Hep G2 cells and comparison with acute human toxicity data.

Within the framework of the EDIT (Evaluation guided Development of In vitro Toxicity and toxicokinetic tests) programme, the long-term cytotoxicity of 27 chemicals was investigated on Hep G2 cells. The first step in the experiments was to determine the PI50(24h) of the chemicals. This is the concentration of compound needed to reduce the total protein content by 50% after 24 h of treatment. In the long-term experiments the chemicals were tested in six different concentrations, using the PI50(24h) as maximum concentration. The cells were treated twice a week with the same concentration of test compound and were trypsinised and counted once a week (dynamic culture). The number of cells was compared to the number of cells of the control. Three major long-term cytotoxicity patterns could be distinguished. After 6 weeks, the EC50(6w)s were determined. This is the concentration of compound needed to reduce the number of cells by 50% after 6 weeks of treatment. These values were compared with the PI50(24h). A good correlation was found for the 27 chemicals (r(2)=0.860). After 6 weeks, the concentration of test compound needed to reduce the total cell protein content by 50% after 24 h after 6 weeks of pretreatment of the cells with a particular concentration of test compound was measured: the PI50(24h-6w). For the majority of compounds there is no difference between the PI50(24h) and the PI50(24h-6w). For ethanol, arsenic (III) oxide, verapamil hydrochloride and orphenadrine, the PI50(24h-6w) increased in comparison to the PI50(24h). For some compounds a doseresponse was observed, indicating that the cells have become more resistant or more sensitive. Linear regression analysis revealed a good correlation (r(2)=0.709) between the EC50(6w) and the human acute toxicity. All these data indicate that a good alternative test may be found for predicting the long-term human toxicity.

Cytotoxicity of the MEIC reference chemicals in rat hepatoma-derived Fa32 cells.

The cytotoxicity of the MEIC reference chemicals was investigated in rat hepatoma-derived Fa32 cells. The total protein content was measured as an endpoint after exposure times of 30 min and 24 h, both in normal and glutathione-depleted cells. The neutral red uptake inhibition and the MTT conversion were also measured after 30 min. On average, the cytotoxicity was higher in glutathione-depleted cells when compared to normal cells, and was lower after 30 min than after 24 h. Evidence was obtained for lysosomal attack (of five chemicals) or mitochondrial dysfunction (of six chemicals) as the primary intoxication mechanism. Malathion and mercuric chloride belong to both series of chemicals. Good to excellent correlations were observed when the 50% inhibitory concentrations of the six different in vitro assays were compared. When the six in vitro assays in Fa32 cells were compared with the human toxicity, the correlation coefficient was almost always identical to that obtained previously in human hepatoma-derived Hep G2 cells. The latter was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Altogether the results integrate very well with the basal cytotoxicity concept (Ekwall, B., 1995. The basal cytotoxicity concept. In: Goldberg, A.M., Van Zutphen, L.F.M. (Eds.), The World Congress on Alternatives and Animal Use in the Life Sciences: Education, Research, Testing. Mary Ann Liebert Publishers, New York, pp. 721-725).

Toxicological Evaluation of Surface-water Samples in Sensitised Cultured Fish Cells Compared with the Microtox® Method.

A previously described neutral red uptake-inhibition assay on cultured fathead minnow (FHM) fish cells revealed a good correlation with fish toxicity data. The assay was made more sensitive by reducing the number of cells, by using a longer treatment period, and by simultaneous treatment of the cells with sodium dodecyl sulphate and L-buthionine (S,R)-sulphoximine. The fluorimetrically quantified protein content was then used as the endpoint. The results were compared with toxicity data obtained by the Microtox® method with the bacterium, Vibrio fischeri. A series of 82 surface-water samples were investigated. Cell quantitydependent and sample concentration-dependent reductions in total protein content were observed. In all, 32 samples were toxic in the FHM assay. Of the seven samples that were shown to be toxic in the Microtox assay, only three were also toxic in FHM cells. No linear relationship was found for the toxicity results obtained with the two methods. Further research to explain the significance of the many positive responses and to identify possible confounding factors will increase the reliability of the sensitised FHM cell assay.

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

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 1 h. 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-buthionine 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 endogenous GSH content after 24 h. t-BHP and H2O2, but not 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. Since H2O2 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.

Glutathione-dependent cytotoxicity of the chloroacetanilide herbicides alachlor, metolachlor, and propachlor in rat and human hepatoma-derived cultured cells.

Alachlor, metolachlor, and propachlor are widely used chloroacetanilide herbicides. Their cytotoxicity in rat (Fa32) and human (Hep G2) hepatoma-derived cells was investigated, in connection with their influence on the endogenous glutathione (GSH) content, on the xenobiotic-metabolizing phase I enzymes 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD), and phase II glutathione transferase (GST). The cytotoxicity was measured by the neutral red uptake inhibition assay. The following toxicity range was observed in both cell lines: propachlor > alachlor > metolachlor. When the endogenous GSH content was reduced by pretreatment of the cells with L-buthionine (S,R)-sulfoximine, the cytotoxicity of the herbicides increased strongly in both cell lines. EROD and PROD activities were dose-dependently increased to different degrees in Fa32, as was EROD in Hep G2, but no PROD activity was observed in these cells. The GSH content was not altered after 1 h treatment, and was approximately doubled after 24 h. GST activity was increased in Fa32 cells but not in Hep G2. A comparable cytotoxicity was observed for the investigated chloroacetanilides in both the rat and the human cell lines. Different interactions with xenobiotic-metabolizing phase I and II enzymes were observed, and GSH showed a protective effect against the acetanilides in both cell lines.

CYP1/2 Activation and Glutathione-dependent Cytotoxicity of Four Pesticides in Hep G2 and Fa32 Cells.

The cytotoxicity of the carbamate insecticide carbaryl, the organophosphate insecticide quinalphos, the benzimidazole fungicide benomyl, and its debutylcarbamoylated derivative carbendazim was investigated in rat (Fa32) and human (Hep G2) hepatoma-derived established cell lines. Benomyl was the most toxic of the four pesticides, followed by quinalphos, carbaryl and the least toxic carbendazim, suggesting that the butylcarbamoyl group plays an important role in the toxicity of benomyl. EROD (7-ethoxyresorufin-O-deethylase) and PROD (7-pentoxyresorufin-O-depentylase) activity were moderately activated in Fa32 cells. In Hep G2 cells no PROD was measured, but EROD was activated 2.5 to 28 times the control values. Piperonyl butoxide and 1-aminobenzotriazole did not influence the cytotoxicity of the pesticides. However, when the endogenous glutathione content was reduced by pretreatment of the cells with l-buthionine-S,R-sulfoximine, the cytotoxicity of the pesticides strongly increased in both cell lines. In conclusion, the same cytotoxicity was observed for the four pesticides in both the animal and the human cell line. CYP1/2-dependent enzymes were activated to different degrees. No evidence was obtained for a cytochrome P450-dependent cytotoxicity, but glutathione showed a protective effect against the four pesticides in both cell lines.

Increased cytotoxic sensitivity of cultured FHM fish cells by simultaneous treatment with sodium dodecyl sulfate and buthionine sulfoximine.

A previously described neutral red uptake inhibition assay on cultured fathead minnow (FHM) fish cells revealed a good correlation with fish toxicity data (Brandao et al., 1992) for a series of 50 test compounds, belonging to completely different chemical classes. The major drawback was the lower sensitivity of the cytotoxicity assay. Aiming at a higher sensitivity the assay was adapted by reducing the cell number, by a longer treatment period, and by simultaneous treatment with sodium dodecyl sulfate and buthionine sulfoximine (BSO). The fluorimetrically measured protein content was chosen as the endpoint. The endogenous glutathione (GSH) content was reduced by 54% of the test chemicals, confirming the importance of GSH in detoxification processes. Higher sensitivity was, at least partly, obtained by treating the FHM cells with BSO, reducing the GSH content to 22% of that in control cells. The cytotoxicity of the 50 chemicals was measured using the modified more sensitive assay. Although some exceptions were observed, this new assay is at least one order of magnitude more sensitive. Toxicity values comparable with fish toxicity data were obtained, making the assay sensitive enough to measure the toxicity of environmental water samples.

Activation by sodium fluoride of drug-metabolizing enzymes in rat hepatoma-derived Fa32 cells.

Protection against xenobiotic insult, including cancer chemoprotection, can be achieved by a variety of natural and synthetic compounds belonging to over 20 different classes of chemicals. They all induce or activate drug-metabolizing enzymes. The discovery of a new class of activator is currently reported. Sodium fluoride activated the phase I ethoxyresorufin-O-deethylase (to 240%) and pentoxyresorufin-O-depentylase (to 156%), and the phase II glutathione transferase to 120% of the basal activities in rat hepatoma-derived Fa32 cells. It is, therefore, a bifunctional enzyme activator. A time- and concentration-dependent activation was observed. A possible impact of the daily fluoride uptake from drinking water is suggested.

Comparative cytotoxicity of 5-aminosalicylic acid (mesalazine) and related compounds in different cell lines.

Nonsteroidal anti-inflammatory drugs can cause serious side-effects such as tubulo-interstitial nephritis. Mesalazine (5-ASA, 5-aminosalicylic acid) is used for the treatment of colitis ulcerosa, Crohn disease, and other diseases; it has been found to induce necrosis of both proximal convoluted tubules and renal papillaries. The comparative cytotoxicity of 3-, 4-, and 5-aminosalicylic acid, acetylsalicylic acid (AcSA), and the parent compound salicylic acid (SA) was investigated for the free acids and for their sodium salts. The interaction with endogenous glutathione (GSH) was also investigated. Four established cell lines were used: MDCK, LLC-PK1, NRK as renal cells, and HepG2 as hepatic cells. The free acid compounds were less toxic than their corresponding salts. Acidic 5-ASA was the most toxic of the three isomers in MDCK and LLC-PK1 cells, while NRK and HepG2 were more susceptible to acidic 3-ASA. Addition of NaOH modified the relative toxicity of 3-ASA and 5-ASA. The LLC-PK1 and HepG2 cells were more sensitive to the test chemicals as their salts than were the NRK and MDCK cells. SA and 5-ASA decreased the GSH content in renal cells and increased it in HepG2. GSH depletion with L-buthionine-(S,R)-sulfoximine enhanced the toxicity only for SA in NRK and for 5-ASA and AcSA in HepG2. No correlation between endogenous GSH and the susceptibility of MDCK and LLC-PK1 to the test compounds was observed. The results suggest that no typical nephrotoxic effect occurred. No explanation could be found for the tubulo-interstitial nephritis caused by 5-ASA therapy.

Purification and characterisation of the soluble glutathione S-transferase isoenzymes in rat kidney derived NRK cells.

Glutathione S-transferase (GST) enzymes are toxicologically important from many points of view. Rat kidney derived established NRK cells were mass cultured for the isolation of GST isoenzymes. These were enriched by affinity chromatography and separated by chromatofocusing and HPLC. Exactly the same major GST subunits were found in NRK cells as in the rat kidney. Strong evidence was also found for the presence of an aberrant form of GST 7-7, as was described in rat kidney. A very good correlation between the NRK GST and rat kidney, and especially cis-platinum treated kidney, was found. It is concluded that NRK cells can be considered as a valuable alternative tool for in vitro research of rat kidney phenomena, especially when toxicological interactions are investigated.

Isolation and characterisation of the class alpha, mu and pi glutathione transferases in LLC-PK1 and pig kidney.

Glutathione S-transferase (GST) isoenzymes from pig kidney cortex and LLC-PK1 (an established cell line derived from the pig proximal tubule) were purified by affinity chromatography, anionic and cationic chromatofocusing. Purification revealed nine isoenzymes in the pig kidney cortex and five isoenzymes in the LLC-PK1 cell line. SDS-polyacrylamide gel electrophoresis showed that the pig kidney cortex isoenzymes were homo- or heterodimeric; LLC-PK1 isoenzymes, however, were homodimeric. Isoenzymes from pig and LLC-PK1 showed a higher affinity towards glutathione. The isoenzymes were further characterised and divided into the different GST classes by studying specific inhibitors, specific substrates and immunological properties. Pig GSTs belong to class alpha, mu and pi. The GSTs in LLC-PK1 cells, on the other hand, belong to class pi and mu. The isoenzyme pattern in LLC-PK1 cells indicates the dedifferentiation of this particular cell line compared with the pig kidney cortex.

The cytosolic glutathione S-transferase isoenzymes in the dog kidney cortex as compared with the corresponding MDCK renal cell line.

Cytosolic glutathione S-transferase (GST) (EC 2.5.1.18) isoenzymes of dog kidney and MDCK (an established dog renal cell line) were purified and studied. Specific GST activity was 248 and 317 nmol/min/mg protein, for dog and MDCK, respectively. Cytosolic GST was only partially purified by glutathione affinity chromatography, a substantial amount (43% and 84% for dog kidney and MDCK, respectively) of the GST activity was found in the flow-through fraction. Affinity bound GST was separated into 6 and 3 isoenzymes by anionic chromatofocusing for dog and MDCK, respectively. Flow-through GST was purified by gel filtration, anion exchange chromatography and anionic chromatofocusing showing only one GST isoenzyme, with distinct features from the affinity bound GST, for both dog and MDCK. The isoenzymes were characterized by their kinetic properties, subunit composition, specific substrates and inhibitors and immunoblot. The major dog GSTs (DII, DIV and DVI) correspond to the MDCK isoenzymes (MI, MII and MIII). Comparable pI values, a comparable affinity towards GSH and comparable sensitivities towards the inhibitors N-ethylmaleimide (NEM), triphenyltin chloride, cibacron blue and hematin were observed for the corresponding isoenzymes: DII and MI, DIV and MII, DVI and MIII. Co-electrophoresis showed that the subunit composition was identical for DII and MI, and for DIV and MII. Inhibitor and substrate sensitivities showed that the affinity bound GSTs belong to class pi and mu, the presence of class pi was confirmed by immunoblot analysis. One homodimeric GST isoenzyme was observed in the dog kidney and MDCK flow-through. Both dog and MDCK isoenzyme have a nearly neutral pI, a high affinity towards CDNB and an equal sensitivity towards triphenyltin chloride, cibacron blue and hematin. However, based on inhibitor studies and immunoblot, this isoenzyme could not be attributed to an identified GST class. The overall isoenzyme pattern of dog and MDCK affinity bound and flow through GST is comparable. The dog and MDCK affinity bound GSTs have similar characteristics and all belong to class mu or pi.