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.

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.

The influence of picolines on glutathione transferase activity and subunit composition in human liver derived Hep G2 cells.

Hep G2 cells, an established cell line derived from a human hepatoma, have retained a number of hepatocytic phase I and II reactions. The influence of picolines (2-, 3- and 4-methylpyridine), related compounds and some classical enzyme inducers on specific glutathione transferase (GST) activity and its subunit composition in Hep G2 cells was investigated. Increased GST activity was observed for rifamycin, phenobarbital, pyrazine and the picolines, of which the 4-isomer was the strongest inducer. The GST subunits were analysed by HPLC. GSTP1, GSTM1a, GSTA1 and GSTA2 were present in control Hep G2 cells. GSTM1a disappeared or was strongly reduced under the influence of the test chemicals. All GST increases were due to augmented GSTA1 expression. Thus, picolines stimulate GST activity in Hep G2 cells by influencing the class alpha GSTA1.

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).

Urinary gamma-glutamyl transferase as a specific marker for mercury after heavy metal treatment of rats.

Male Sprague--Dawley rats were injected i.p. with 1 mg heavy metal (Cu, Rb, Cs, Zn, Sr, Cd, Ba, Zr, Pb, Mb, Fe, Co, Ni)/kg body weight, as chlorides and 24 h urine samples were analysed for gamma-glutamyl transferase (GGT) content. Mercury (Hg) was the only metal that induced an enhanced urinary GGT activity. We concluded that, when acute metal intoxication has been observed, urinary GGT may function as a specific marker for Hg intoxication. Gel permeation studies showed that increased tubular lesion accounts for the Hg-induced GGT increase.

Soluble glutathione S-transferase isoenzymes in rat brain.

The soluble glutathione S-transferase (GST) isoenzymes in rat brain were investigated using 1-chloro-2,4-dinitrobenzene (CDNB) as the second substrate. The percentages of the different CDNB-GST isoenzymes found were: anionic GST: 3.7%, GST D + E: 35.3%, GST C: 27.9%, GST B: 0.5%, GST A: 13.9% and GST AA: 18.6%. The percentages of isoenzymes are quite different from those measured in liver, testis and prostate. An important detoxication role of GST in brain is suggested.

Prostatic glutathione S-transferase in rat, guinea pig and rabbit.

The occurrence of soluble glutathione S-transferase (GST) in the prostate of rat, guinea pig and rabbit is reported. With 1-chloro-2,4-dinitrobenzene (CDNB) as the second substrate a specific activity of 59 nmol product/min/mg protein was found in the rat. A value of approx. 2 x and 25 x higher was found for guinea-pig and rabbit respectively. GST activity was also demonstrated with 3 other substrates. GST C was the most abundant CDNB-GST isoenzyme (76%) in rat prostate, followed by GST A (17%) and GST AA (7%), significant amounts of other isoenzymes not being found. The presence of GST in the prostate suggests that these enzymes may play a role in the metabolism and detoxification of electrophilic xenobiotics.

Gel filtration of urine: a method to detect nephrotoxicity in rats.

Sephadex G-100 gel filtration of urine from male Wistar rats revealed 3 protein peaks: peak I (eluted with the void volume of the column), peak II (Mr between 67 000 and 43 000), and peak III (Mr about 13 700). Nephrotoxic compounds were given as a single i.p. injection. Peak I, and especially peak II were significantly increased in 24-h urine samples from rats receiving mercuric acetate (1.58 mg/kg), mercuric trifluoroacetate (MTFA) (2.22 mg/kg), sodium ethylmercurithiosalicylate (EMTSA) (20.2 mg/kg), sodium tetrathionate (250 mg/kg), ammonium fluoride (18.5 mg/kg), paramomycin sulfate (800 mg/kg), ochratoxin A (5 mg/kg) and cis-platinum (4 mg/kg). It is concluded that gel filtration of urine can be used as a method to detect nephrotoxicity in rats.

Indications for the presence of sulfate esters as considerable metabolites of sunset yellow FCF and orange GGN in rat faeces.

Male Wistar rats received by stomach intubation 500 mg/kg of the azo dyes Sunset Yellow FCF and Orange GGN. Previous work revealed that in the Sunset Yellow FCF as well as in the Orange GGN aqueous faeces extracts a considerable orange metabolite is present, eluting as a strong peak before the unchanged dye after ion-pair reverse-phase liquid chromatography (RP-IP-HPLC). Incubation of both aqueous faeces extracts with arylsulphatase followed by high-performance liquid chromatography (HPLC) analysis, resulted in a significant orange metabolite peak height decrease together with a noticeable unchanged dye peak height increase. By means of semi-preparative HPLC, combined with cation-exchange purification, the orange Sunset Yellow FCF faeces metabolite could be isolated. It was identified by means of FT-1HMR spectroscopy as a Sunset Yellow FCF structurally related compound with the same 1HMR pattern, which is therefore modified on the free aromatic hydroxyl group.

Mutagenic activation of aromatic amines by a human hepatoma cell (Hep G2) supernatant tested by means of Salmonella typhimurium strains with different acetyltransferase activities.

The study was carried out to characterize hepatoma cells (Hep G2) as activation system relevant to man and to investigate which are the tester strains most suitable for the mutagenic assay of aromatic amines. A supernatant prepared from the human hepatoma cell line Hep G2 was used to activate benzidine, 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) in the Salmonella typhimurium reversion assay. Activation by Hep G2 supernatant was studied with increasing concentrations of the three compounds, in tester strains TA98, YG1024, DJ400 and DJ460. Benz[alpha]anthracene (BA) pretreatment of cells increases the mutagenicity of benzidine in strains YG1024, DJ460 and DJ400. Activation of 2-AAF and 2-AF was observed in strains YG1024, DJ400 and, at the highest tested dose, in DJ460. These results were compared with those obtained with S9 from control and Aroclor 1254 (Aro)-pretreated rat liver. With strain TA98 comparable responses were obtained except for 2-AF which was better activated using rat liver S9. The use of strain YG1024 greatly increases the sensitivity of the response. Strain DJ460 makes it possible to detect activation of 2-AF and 2-AAF by Aro-induced rat liver. These results indicate that Hep G2 supernatant is a useful metabolic activation system of human origin that can be used to replace rat liver S9. An appropriate choice of the Salmonella strain not only can increase the sensitivity of the response, but may also help to overcome certain metabolic shortcomings of the Hep G2 cell line and rat liver S9.

Metabolic activation by a supernatant from human hepatoma cells: a possible alternative in mutagenic tests.

The supernatant from human Hep G2 hepatoma cells was examined for typical enzymatic activities involved in the metabolism of xenobiotics. Neither cytochrome P-450 nor b5 was detectable, but associated enzymatic activities were found especially after induction with hydrocortisone (HC) and benzanthracene (BA) suggesting that this Hep G2 supernatant contains cyt P-450 IA1 and IA2. Other critical enzymes are also present, but, as expected, at lower activities than in Aroclor 1254 rat liver S9, except for NADH and NADPH cytochrome c reductase. Results of the Ames test indicate that the induced Hep G2 supernatant is a suitable activator for the evaluation of genotoxicity of indirect mutagens.

Correlation between the reduction of protein content in cultured FHM fish cells and fish lethality data.

The reduction of total protein content in a fish-derived cell line was investigated as an alternative method for the assessment of some ecotoxicological properties of compounds. The 25 chemicals tested included various inorganic compounds and organic compounds such as alcohols, acids, ketones, etc. The xenobiotics were applied at different concentrations to cultured fathead minnow (FHM) fish cells. After 2 hr contact time the reduction of the total protein content was measured. The results are quantified by the PI(50) value, which is the concentration of test compound required to induce a 50% reduction in total protein content. The PI(50) values were compared with fish lethality data (LC(50)) obtained in golden orfe and a correlation coefficient (r = 0.90) was found. The reducton in total protein content in FHM fish cell cultures correlates very well with lethality data determined on fish, is easy to perform, and can be considered as a good alternative in vitro assay for ecotoxicity testing of a wide variety of chemicals.

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.

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.

Cytotoxicity testing of 114 compounds by the determination of the protein content in Hep G2 cell cultures.

The cellular protein content measured in cultured Hep G2 cells was used as the endpoint for determining the cytotoxicity of a range of 114 chemical compounds. The relative toxicity of the test compounds was quantified by the determination of the PI(50), which is the concentration of xenobiotic required to produce a 50% reduction in protein content of the culture after 24 hr. Surfactants and heavy metals consistently had low PI(50) values. Hep G2 cells were very sensitive to compounds with more than one carboxyl group. Triacetin and glutathione were identified as false positives. The results suggest that the PI(50) assay could be a useful pre-screening method to test for the cytotoxicity of chemicals.

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.

Effects of foetal calf serum on cell viability, cytotoxicity and detoxification in the two kidney-derived cell lines LLC-PK1 and MDCK.

Foetal calf serum (FCS) dependent cell viability, cytotoxicity and detoxification were investigated in MDCK and LLC-PK1 cells. FCS was used at 0-10% (v/v). Viability and cytotoxicity were measured by neutral red uptake and by the MTT test. Viability of LLC-PK1 was strongly dependent on the FCS concentration, but that of MDCK cells only to a very limited extent. For both cell lines the cytotoxicity of HgCl(2) was FCS concentration dependent: lower toxicity was observed with 5-10% FCS than with 0-1% FCS. This effect was not observed for paracetamol. The results could not be explained by altered glutathione or glutathione S-transferase. The optimal FCS concentration of 1%, necessary to retain cell viability, had a limited influence on cytotoxicity. FCS concentration must be taken into consideration when cytotoxicity data from different studies are compared.

Interaction of chlorophenoxyalkyl acid herbicides with rat-liver glutathione S-transferases.

The in vitro interaction of four chlorophenoxyalkyl (CPA) acid herbicides with rat-liver glutathione S-transferase (GST) was studied using reduced glutathione and 1-chloro-2,4-dinitrobenzene as substrates. Inhibition of GST activity by the CPA acids in crude extracts was dose dependent. Ring substitution and side-chain length were shown to be of importance in determining the extent of GST inhibition. While GST AA, an isoenzyme of GST, was stimulated by two CPA acids, each of the other GST isoenzymes (A, B, C, E and M) was inhibited, to different degrees. Kinetic studies revealed a mixed type inhibition of the isoenzymes. Conjugates of CPA acids with glutathione were not formed. These results indicate that CPA acids interact with GST by binding directly to these proteins, possibly at a different locus from that of the substrate. The binding of CPA acids to GST may, therefore, have a protective function against these herbicides.

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.

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.