gamma-Glutamyl transpeptidase catalyses the extracellular detoxification of cisplatin in a human cell line derived from the proximal convoluted tubule of the kidney.

Nephrotoxicity is a side-effect and the main factor limiting the clinical use of cisplatin. In vivo, the administration of the cysteine-containing tripeptide glutathione (GSH) has been found to reduce nephrotoxicity, but the biochemical mechanism of this protective action is not fully understood. The present study was designed to gain insights into the mechanism by which GSH prevents cisplatin nephrotoxicity. We also wanted to verify the hypothesis of whether the protective action of GSH is mediated by products of the extracellular breakdown of GSH catalysed by gamma-glutamyl transpeptidase (GGT), an enzyme that is highly expressed in kidney tubular cells. The study was performed in HK-2 cells, derived from the immortalisation of human kidney proximal tubule cells. We investigated the influence of modulators of GGT activity and/or thiols on the antiproliferative activity of cisplatin and on the intracellular GSH content. We determined the antiproliferative activity of cisplatin, platinum cellular accumulation and DNA platination following precomplexing of the drug with thiols. The antiproliferative effect of cisplatin was minimally affected by the addition of GSH. However, when the antiproliferative assay was performed in the presence of glycyl-glycine (GlyGly), to serve as a transpeptidation acceptor and thus to stimulate GGT-mediated GSH catabolism, cisplatin-induced growth inhibition was largely prevented. This effect was not mediated through an increase of intracellular GSH levels, which were not affected by the GlyGly supplementation. The thiol dipeptide cysteinyl-glycine, i.e. the GSH catabolite generated by GGT activity, showed a higher reactivity against cisplatin in vitro than GSH, as was shown by the more rapid oxidation of its -SH groups. The cisplatin/GSH or cisplatin/cysteinyl-glycine adducts did not display an antiproliferative effect. However, 2 h precomplexing with GSH in the presence of GGT, or directly with the GSH catabolite cysteinyl-glycine, decreased the antiproliferative effect of cisplatin and drug-induced DNA platination to a greater extent than precomplexing with GSH alone. The results of the present study show that, in HK-2 cells, extracellular GSH decreases the antiproliferative effects of cisplatin only upon its hydrolysis by GGT, thereby supporting the hypothesis that the extracellular metabolism of GSH by GGT plays a role in modulating cisplatin nephrotoxicity. A primary role in the protection of HK-2 cells appears to be played by cysteinyl-glycine, the proximal product of the GGT-mediated hydrolysis of GSH, which shows a high reactivity against CDDP resulting in the rapid inactivation of the drug.

P-glycoprotein in HK-2 proximal tubule cell line.

P-glycoprotein (PGP) is an efflux pump physiologically expressed in the apical membrane of the proximal tubular cells. PGP may play a role in the elimination of exogenous substances such as chemotherapeutic drugs, calcium channel blockers and immunosuppressors. The involvement of renal PGP in the transport of endogenous substrates is under investigation. HK-2 is an immortalized proximal tubule cell line from normal adult human kidney, reported to retain a phenotype indicative of a well-differentiated state. No data regarding expression and/or activity of PGP in this cell line are available. The aim of this study was to ascertain the usefulness of HK-2 cell line to investigate the properties and roles of PGP in proximal tubular cells. PGP expression in HK-2 cells was determined by immunoblotting analysis using the monoclonal antibody C219. The activity of PGP was assessed by measuring the transport of the fluorescent probe Rhodamine 123 (R-123) in intact cell monostrates. The interactions of putative PGP modulators, including verapamil and cyclosporin A were also evaluated. Western blot revealed a C219 immunoreactive band of about 150 kDa consistent with the presence of PGP. HK-2 cells preloaded with R-123 rapidly effluxed the dye, the efflux being inhibited by verapamil. Verapamil and, to a major extent cyclosporin A, significantly increased R-123 intracellular accumulation. PGP immunoblottable amount was increased when cells were cultured in the presence of either cyclosporin A or dexamethasone. The results suggest that the HK-2 cells, among the various differentiation features of proximal tubules, retain also the expression of a functional PGP in their membranes and that both PGP activity and expression may be modulated by drugs. Therefore, HK-2 line appears a suitable and promising tool for the study in vitro of renal transport processes dependent on PGP.

Effects of curcumin on P-glycoprotein in primary cultures of rat hepatocytes.

Curcumin is a natural phenolic compound found in the rhizomes of Curcuma longa and endowed with beneficial biological activities including antioxidant, anticarcinogenic and hepatoprotective effects. In this study curcumin was tested for its potential ability to interact in vitro with hepatic P-glycoprotein (Pgp), in a model system represented by primary cultures of rat hepatocytes, in which spontaneous overexpression of multidrug resistance (mdr) genes occurs. In both freshly-plated hepatocytes, containing low levels of Pgp, and 72 hour-cultured hepatocytes, containing high levels of Pgp, the Rhodamine-123 (R-123) efflux, which represents a specific functional test for Pgp-mediated transport, was inhibited by curcumin in a dose-dependent manner. Western blot analysis showed that 25microM curcumin, when included in the culture medium throughout the experimental observation (72 hours), was able to significantly lower the increase of mAb C219-immunoreactive protein spontaneously occurring in the cells during culture. Curcumin, at doses ranging from 50 to 150microM was cytotoxic for freshly-plated hepatocytes, as shown by the strong decrease in the cell ability to exclude trypan blue 24 hours later, but it was significantly less cytotoxic when added to 24 or 48 hour-cultured cells. The resistance to curcumin, progressively acquired by cells during culture, was significantly reduced by high concentrations of dexamethasone (DEX) or dimethyl-sulfoxide (DMSO), culture conditions known to inhibit the spontaneous overexpression of Pgp. In addition, in a concentration-dependent manner, verapamil reverted curcumin resistance in Pgp overexpressing hepatocytes. In photoaffinity labeling studies, curcumin competed with azidopine for binding to Pgp, suggesting a direct interaction with glycoprotein. These results suggest that curcumin is able to modulate in vitro both expression and function of hepatic Pgp and support the hypothesis that curcumin, a chemopreventive phytochemical, could reveal itself also as a compound endowed with chemosensitizing properties on mdr phenotype.

Localization of a GSH-dependent dehydroascorbate reductase in rat tissues and subcellular fractions.

A novel GSH-dependent dehydroascorbate (DHA) reductase from rat liver cytosol has been recently purified and partially characterized in our laboratory. A further characterization study has been carried out in order to determine intracellular and tissue distribution of the enzyme. A modified purification method, yielding a threefold increase in enzyme activity recovery, has been used. Polyclonal antibodies were obtained in rabbits and specific anti-DHA reductase IgG were purified by affinity chromatography employing the homogeneous enzyme as ligand. Immunoblotting analysis of subcellular fractions showed the exclusively cytosolic location of the enzyme. Immunotitration experiments, performed in order to determine the percentage of cytosolic DHA reductase activity ascribable to our enzyme, revealed that purified enzyme activity was completely titrable, while only 70% of DHA reducing activity was titrable in liver cytosol preparation. When immunoblotting analysis was employed to determine tissue distribution of the enzyme, liver, intestinal mucosa, kidney, adrenals, submaxillary gland, testis, and pancreas appeared most endowed with the enzyme, and lower levels were observed in all the other tissues examined. Immunohistochemical studies showed clear zonal distributions in kidney and intestinal tract and overall homogeneous patterns in the other tissues.

Hepatotoxicity and P-4502E1-dependent metabolic oxidation of N,N-dimethylformamide in rats and mice.

A comparative biochemical and histological study on the hepatotoxicity of a single dose of N,N-dimethylformamide (DMF) and N-methylformamide (NMF) in control and acetone-treated SD male rats and CD-1 male mice was performed. In control and acetone-pretreated rats, neither DMF nor NMF caused hepatic damage or elevation of plasma transaminases. In contrast, in acetonized but not in control mice, DMF administration yielded some evidence of liver necrosis and elevation of ALAT (alanine-amino transferase) activity. After a DMF dose of 1000 mg/kg, ALAT activity was found 1215 +/- 832 mU/ml and 47 +/- 18 mU/ml in acetonized and control mice, respectively. NMF treatment was hepatotoxic in control mice and lethal in acetonized mice. In control mice, an NMF dose of 600 mg/kg increased ALAT activity from a basal value of 35 +/- 5 to 2210 +/- 1898 mU/ml. When the oxidative metabolism of DMF was investigated, microsomes from both rats and mice preinduced by acetone increased the demethylation rate of DMF 7 to 10-fold compared to that (about 0.25 nmol/min per mg protein) of the corresponding control microsomes. The enzymatic affinities for DMF oxidation, however, were different: in mice the Km (0.05 mM) was one order of magnitude lower than that (0.56 mM) found in rats. The experiments performed with purified rat and mouse P-450 2E1 in a reconstituted system confirmed that the P-450 2E1 isoforms are very active catalysts towards DMF oxidation (the turnover was about 10 nmol/min per nmol P-450 for both enzymes) but with a strikingly different affinity. Whereas the Km for mouse P-450 2E1 was 0.08 +/- 0.03 mM, that for rat P-450 2E1 was 1.1 +/- 0.2 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of flavonols on P-glycoprotein activity in cultured rat hepatocytes.

The effects of flavonols on P-glycoprotein (Pgp) activity were studied in cultured rat hepatocytes by assessing and transmembrane transport of Rhodamine-123 (R-123) and doxorubicin (DOX). In freshly-plated hepatocytes, containing a low amount of Pgp, flavonols did not affect the cellular retention of DOX, but strongly inhibited the Pgp-mediated efflux of R-123. In 72h-cultured hepatocytes, spontaneously overexpressing functional Pgp, flavonols inhibited R-123 efflux in a dose-dependent manner, but significantly reduced DOX retention while increasing its efflux. A similar effect was found in hepatocytes obtained from rats in which Pgp was induced in vivo by 2-acetamino-fluorene (AAF) or alpha-naphthyl-isothiocyanate (ANIT) treatments. These findings indicate that flavonols, dietary compounds reported to strongly upregulate the apparent activity of Pgp in cancer cell lines, may also modulate differently the transport of putative Pgp substrates in normal rat hepatocytes. The ability to affect the drug-extruding activity at the hepatocyte canalicular membrane could be of relevance to the chemopreventive action of these compounds towards liver carcinogens.

Influence of rat strain on P-glycoprotein expression in cultured hepatocytes.

The amount and activity of the multi-drug transporter P-glycoprotein (Pgp) have been measured in cultured hepatocytes derived from different rat strains. A marked increase in Pgp, as revealed by Western blotting, occurred 48 h after seeding in hepatocytes from Sprague-Dawley, Wistar and Fischer 344 rats, the last showing the highest value. The addition of dexamethasone (DEX) to culture medium delayed Pgp overexpression in all the strains, proportionally to the protein amount in the absence of hormone. The R-123 functional test for Pgp showed that Fischer 344 hepatocytes had the lowest ability to extrude the fluorescent dye as compared with Sprague-Dawley or Wistar rats. These results suggest that the Fischer 344 rat is more prone than other strains to culture stressing conditions, leading to an overexpression of Pgp that is not necessarily functional.

Differential modulation of P-glycoprotein expression by dexamethasone and 3-methylcholanthrene in rat hepatocyte primary cultures.

Spontaneous and culture condition-dependent changes in P-glycoprotein expression and activity have been monitored in primary cultures of rat hepatocytes by using immunoblotting, PCR and fluorimetric techniques. In hepatocytes cultured in basal medium without addition of dexamethasone or 3-methylcholanthrene, mdr mRNA and P-glycoprotein increased progressively throughout a 72 h culture period, in concert with an enhancement in the ability to extrude the fluorescent dye Rhodamine-123. Addition of 1 microM dexamethasone to the culture medium slowed down the increase in mdr mRNA and P-glycoprotein, while inducing a significant increase in the efficiency of R-123 efflux. Addition of either 100 nM or 10 microM DEX produced different changes in mdr mRNA and protein, unrelated to the rate of Rhodamine-123 extrusion. When 50 microM 3-methylcholanthrene was added to the culture medium in the absence of any hormone supplementation, no significant changes in P-glycoprotein activity and expression took place, in comparison with control cultures. On the contrary, in the presence of dexamethasone (100 nM and 1 microM), 3-methylcholanthrene induced an increase in mdr mRNA and in the amount of immunoblottable protein during culture, without producing any concomitant increase in the efficiency to extrude Rhodamine-123. The last phenomenon resulted to be an artefact, since 3-methylcholanthrene was shown to inhibit Rhodamine-123 transport competitively. These results indicate that rat hepatocyte P-glycoprotein may be variously modulated in vitro, by supplementing culture medium with hormones and/or xenobiotics. Functional activity of the P-glycoprotein is not necessarily related with protein amount and/or mdr RNA.

Assessment of P-glycoprotein-dependent drug transport in isolated rat hepatocytes using rhodamine 123.

To test the activity of P-170 glycoprotein in isolated hepatocytes, a method has been developed employing the fluorescent dye rhodamine 123 (R-123). The uptake of R-123 by both freshly isolated and 4-hr-plated hepatocytes depends on dye concentration, time of incubation, and cell number. The efflux of R-123 from cells is inhibited by sodium azide and by verapamil. In standard conditions the efficiency of efflux of R-123 from cells correlates with the relative amount of immunoblottable glycoprotein. The method has been applied to detection of P-170 activity in hepatocytes from animals of different ages as well as from carcinogen-treated animals. The proposed assay appears a simple and adequate tool for the functional assessment of multidrug transporter in liver.

Inducibility of gamma-glutamyltransferase by dexamethasone in rat liver: relationship with the cytochrome P-450 content.

Gamma-glutamyltransferase (GGT) inducibility by dexamethasone (DEX) in rat liver decreased by about 95% within the first 14 days of life, while the liver content of cytochrome P-450 (P-450) increased by about 500%. Cobaltic Protoporphyrin IX (CPP), given on the 9th day of life, caused a temporary depression of the P-450 liver content, with maximal effects 3 and 4 days after the administration of CPP. GGT induction by DEX was significantly higher in CPP-treated rats than in untreated ones, with maximum induction coinciding with the maximal decrease of P-450. These effects were CPP dose-dependent.

Dexamethasone induction of gamma-glutamyl transferase in primary cultures of hepatocytes is enhanced by metyrapone.

Metyrapone, a cytochrome P-450 inhibitor, reduces by about 3,000 times the dexamethasone concentration required to cause a maximal induction of gamma-glutamyltransferase in adult rat hepatocyte cultures, in itself having no inducing activity. Metyrapone effect decreases as dexamethasone concentration approaches the optimally inducing one. Metyrapone action on low DEX concentrations is dose-dependent while inhibiting 7-ethoxycoumarin O-deethylation by 20% to 75%. At the same doses, metyrapone amplifies also the effects of all the hormonal concentrations inducing tyrosine aminotransferase. These phenomena may be triggered by a modulation of the glucocorticoid biotransformation effective at both transcriptional and translational levels.

Gamma-glutamyltransferase induction by dexamethasone in cytochrome P-450-depleted rat liver.

The effects of the cytochrome P-450 depletion by cobaltic protoporphyrin IX on the postnatal glucocorticoid-inducibility of the membrane-bound enzyme gamma-glutamyltransferase have been assessed in the rat liver. Dexamethasone-induced gamma-glutamyltransferase activity in 14-, 28- and 77-day-old rats was high, weak and absent, respectively, and inversely correlated with the physiological cytochrome P-450 activity. In the liver acinus, the enzyme was reexpressed by the zone 1 and zone 2 hepatocytes in suckling rats, substantially only by the zone 1-hepatocytes in just weaned rats. Following cytochrome P-450 depletion, gamma-glutamyltransferase induction by dexamethasone was more rapid, more intense and more extended in the liver acinus, occurring also in the zone 3 hepatocytes in suckling rats, in the zone 2 and a few zone 3 hepatocytes in just weaned rats. Further, the enzyme induction occurred also in adult rats in the zone 1 and in some zone 2 cells. This shows that cytochrome P-450 modulates the extent of hepatic gamma-glutamyltransferase induction by dexamethasone in postnatal rat-hepatocytes. The phenomenon may be consequent on hormone biotransformation changes caused by the cytochrome P-450 depletion.

Possible role of the acetone-inducible cytochrome P-450IIE1 in the metabolism and hepatotoxicity of thiobenzamide.

The effect of acetone pretreatment (5% in drinking water for 10 days on rat liver metabolism and toxicity of thiobenzamide (TB) was investigated. Hepatic microsomes from acetone-pretreated rats showed a significant increase of TB-S-oxidation rate which, on the basis of selective thermal inactivation of FAD-containing monooxygenase (FADM), appeared dependent only on cytochrome P-450. Furthermore, TB was able to competitively inhibit acetone hydroxylase (AcH), an enzymatic reaction highly specific for the P-450IIE1 isozyme. Acetone pretreatment of rats also produced an exacerbation of liver damage induced by acute administration of TB (150 mg/kg), as judged by the extent of liver necrosis and serum alanine-amino transferase (ALAT) activities. Coadministration of acetone with TB reduced on the other hand the extent of liver damage. The findings suggest that P-450 species induced by acetone, and in particular the P-450IIE1 isozyme, could be involved in the biotransformation of TB.

Changes in the rat liver drug metabolizing system during a short thiobenzamide feeding cycle.

The changes in the hepatic drug metabolizing enzymes induced by the liver tumor promoter thiobenzamide (TB) were investigated. Feeding of TB to rats at a promoting regimen (1 g/kg of diet for 2 weeks) resulted in a significant decrease in the amount of liver microsomal cytochrome P-450 and of total heme. Also, the activity of cytochrome P-450 dependent monooxygenases (aminopyrine demethylase, arylhydrocarbonmonooxygenase and ethoxycoumarindeethylase) and FAD-containing monoxygenase (N,N-dimethylaniline N-oxidase and TB S-oxidase) were depressed. By contrast, phase II enzymes such as epoxide hydrase, UDP-glucuronyl transferase and GSH-transferase were significantly induced. This overall change in the drug metabolizing system was associated with tolerance of the liver towards a high necrogenic dose of TB itself as well as with an increase of mitoses and apoptosis of the hepatocytes. The findings suggest a possible relationship between this TB-induced adaptive response and the promoting activity of the compound on liver carcinogenesis.

Cytological and quantitative cytochemical changes in the hepatocyte population of newborn rats following hydrocortisone administration.

Changes have been assessed in cytological and quantitative cytochemical parameters of the hepatocyte population of newborn rats under glucocorticoid stimulation. Administration of hydrocortisone-acetate at the dose of 25 micrograms/g b.w./d during the 2nd week of postnatal life, caused: 1. an increase of the liver weight and of average dry mass, protein content, and volume of the hepatocytes; 2. a decrease of the number of hepatocytes per mg of liver tissue; 3. a reduction of the mitotic activity in liver parenchyma; 4. a gain in number of hepatocytes per liver lower than under normal conditions; 5. an increase of frequency of binuclear cells; 6. an increase of DNA-Feulgen per hepatocyte nucleus; 7. an increase per cell, greater than the mean protein increase per cell, in activity of arylhydrocarbonmonooxygenase and 7-ethoxycoumarin 0-deethylase, 2 enzymes dependent on cytochrome P-450. Induction of arylhydrocarbonmonooxygenase activity was prevalent in centrolobule. All the examined parameters, except that of DNA-Feulgen per nucleus and that of mitotic activity, changed strictly correlated with the duration of hormonal treatment. The values of a number of hepatocyte parameters (particularly: mean cell dry mass and volume, frequency of binuclear cells, enzymic activity) detected in the 12 d old rats after a 5 d long hormonal pretreatment, were in the range of those of animals 1 to 2 weeks older.

Characterization of the promoting activity of thiobenzamide on liver carcinogenesis.

To characterize the promoting activity of thiobenzamide (TB), a thiono-sulfur-containing xenobiotic, rats of both sexes were treated with the compound for 4 or 8 weeks after an initiating dose of diethyl-nitrosamine. The number, area (volume), and phenotypic complexity of the enzyme-altered hepatocyte foci were studied in serial sections stained with hematoxylin/eosin and reacted for glycogen, iron, and gamma-glutamyltranspeptidase activity. The TB-induced changes on the drug metabolizing systems were also investigated. The main findings were: When fed in a dose range of 500-2,000 mg/kg of diet, TB induced a number of foci greater than controls, especially in female rats. Benzamide, a major TB metabolite, was ineffective. The appearance of hepatocyte foci upon TB feeding was nearly synchronized. An increase of the phenotypic complexity of the hepatocyte foci occurred only during the first 4 weeks of TB administration; it correlated with an increase in size. The liver microsome content of cytochrome P-450 as well as the activity of many monooxygenases was decreased, some of the phase II reactions being increased. In conclusion TB behaves as an efficient promoter of liver carcinogenesis, possibly as a consequence of an induced adaptive response.

Role of the microsomal FAD-containing monooxygenase in the liver toxicity of thioacetamide S-oxide.

To evaluate the different contributions of either microsomal FAD-containing ( FADM ) or cytochrome P-450 dependent monooxygenases in the bioactivation and liver toxicity of thioacetamide-S-oxide ( TASO ) (a proximate metabolite of the liver toxin and carcinogen thioacetamide), this compound: (i) was given to rats pretreated with methimazole (a substrate and inhibitor of FADM ), SKF 525-A (an inhibitor of cytochrome P-450) and cobalt protoporphyrin IX (a synthetic porphyrin which induces a long-lasting depletion of the hepatic cytochrome P-450); and (ii) was added to liver microsomes performing oxidation of model FADM or cytochrome P-450 substrates. Whereas the prior administration of methimazole alleviated the TASO induced liver necrosis, SKF 525-A was almost ineffective. Also pretreatment with cobalt protoporphyrin IX prevented liver necrosis. However, this porphyrin derivative was found to depress both cytochrome P-450 dependent and the FADM dependent biotransformations. On the other hand, addition of TASO to liver microsomes in vitro induced changes in the kinetics of S-oxidation of thiobenzamide and of N-oxidation of dimethylaniline, whereas the O-deethylation of ethoxycoumarin was unchanged. The overall results show the necessity of TASO bioactivation by mixed-function monooxygenases for the toxic action to be apparent; at the same time, the findings suggest FADM as the system mainly involved in TASO metabolism.

Gamma-glutamyltranspeptidase induction by cortisol in liver parenchyma of unweaned rats.

In contrast to many differentiated hepatic functions developing after birth, very little is known about in vivo glucocorticoid influences on postnatal expression of fetal liver enzymes, such as GGT. This study showed that cortisol markedly induces liver GGT activity in unweaned rats, but has no effect after weaning. Enzyme induction was dose- and time-dependent and occurred in parenchymal cells, progressing with time from zone 1 to zone 2 of the liver acinus. Zone-3 hepatocytes were unresponsive even after a 5-day treatment. Lag-times for GGT induction in zones 1 and 2 of the liver acinus were 1 to 2 days and 2 to 3 days, respectively. From this, a permissive cell change, determined by the hormone administration itself, seems required for the hepatocyte GGT induction by cortisol in pre-weaning rats.

Biliary cirrhosis and tumors induced by chronic administration of thiobenzamide to rats.

Thiobenzamide (TB), a thiono-containing compound, was administered for 38 weeks to female Sprague-Dawley rats at a dose of 1 g/kg standard diet; the resulting liver pathology was followed up to 8 months after withdrawal of the compound from the diet. TB administration induced the appearance of biliary cirrhosis. In the first weeks of intoxication the progressive distortion of the liver architecture was mainly due to significant proliferation of the bile ductules. Later, the liver assumed a macronodular appearance. In addition to regenerative and degenerative changes of the hepatocytes, preneoplastic lesions were also detected, and some enzymic markers of the mixed-function monooxygenase system were decreased. Cholangiofibrotic areas were evident, and many biliary tubules within them showed mucous metaplasia. At the end of the intoxication period, as well as 4 months after drug suspension, large portions of the liver or entire lobes were substituted by connective tissue surrounding nests of bile ductules and atrophied hepatocellular nodules. Four months later, in the virtual absence of cirrhotic changes, each animal harboured one or more tumors (mainly cholangiomas).