Gamma-glutamyltransferase induction by glucocorticoids in rat liver: age-dependence, time-dependence, dose-dependence, and intralobular distribution.

Postnatal responsiveness of rat-liver gamma-glutamyltransferase (GGT) to glucocorticoids (GC) has been defined by investigating: age-dependence, time-dependence, hormonal dose-dependence, and lag-time of the enzyme re-expression; half-life of the induced enzyme activity; dynamics of the enzyme reappearance in the liver tissue. Hydrocortisone-acetate (HC) or dexamethasone (DEX) were administered to the animals starting 1, 2, 3, 4, or 5 d before killing, at the doses of 25 micrograms or 1 microgram/(g b. w. x d), respectively. In 14 d old rats, after a lag-time of about 20 h (DEX) or 30 h (HC), GGT activity progressively increased up to 38 and 31 times the control value, respectively, at 5th d; the enzyme re-expression was linearly hormone dose-dependent; half-life of the induced enzyme activity was about 36 h. In 21 d old rats, GGT re-induction behaved as in 14 d old animals, except that the induced activity was about half that of each correspondent treatment. In 28 d old rats, a very low but significant GGT activity was re-expressed only after hormonal treatments longer than 48 h. In 35 and 77 d old rats, significant GGT activity was never re-induced. GGT was re-expressed in liver parenchyma, with a defined space-course. In 14 d old rats, GGT reappeared first in periportal areas, then in acinar zone 1, finally in acinar zone 2. While the animals were ageing, GGT re-expression occurred to lesser and lesser extents in liver tissues, because of a progressive space-restriction from acinar zones 1 and 2 to zone 1 and finally, in 35 d old rats, to periportal areas. In adults, GGT was re-expressed only by rare hepatocytes in periportal spaces. Acinar zone-3 hepatocytes did never re-express GGT, irrespectively of the animal age. Thus, 2 rat hepatocyte populations could be distinguished (1 responsive, the other unresponsive to GC for GGT re-expression), the relative proportion of which changes in favour of the unresponsive one while the animal ages. Hepatic GGT re-induction by GC, occurring after a long lag-time, does not follow the typical model of hormonal induction. Previous permissive cell changes seem to be required. Hepatocyte-GGT re-expression by GC appears to be inversely correlated with the differentiation level and the cytochrome P-450 amount (activity) of the cell as limiting factors for the triggering of the enzyme induction

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

Early biochemical liver changes following thiobenzamide poisoning

Administration of thiobenzamide in a single dose (25 mg/100 g body wt by stomach tube) to male rats induced centrilobular necrosis, which became evident 10 h after the poisoning. In the meantime liver weight and water content underwent changes, glycogen was lost, triglycerides accumulated in the liver while decreasing in serum, [3H]leucine uptake in proteins was impaired and the activity of glucose-6-phosphatase and aminopyrine demethylase decreased. The activity of NADPH-cytochrome c reductase remained unchanged, whereas a reduction of the microsomal cytochrome P-450 occurred. The liver amount of reduced glutathione underwent no significant changes. Pretreatment of the animals with cobalt chloride or 20-methylcholanthrene decreased the liver damage caused by the drug. The in vitro addition of thiobenzamide to liver microsomes resulted in a spectral change. The appearance of conjugated dienes among microsomal lipids from drug-treated rats indicated for a lipoperoxidation taking place in vivo

Circadian rhythm of dry mass and weight-class-pattern of the rat hepatocytes--effects of light-dark and feeding regimens.

1. Dry weight has been determined of individual hepatocytes isolated from rats kept at natural or at reversed daily light-dark cycle, and from rats under time-restricted feeding. Behaviours of liver weight, mitotic activity and binuclearity frequency of the hepatocytes and serum corticosterone have been also investigated. 2. At natural light-dark cycle, liver weight, hepatocyte mitotic activity, and serum corticosterone were higher during the day than during the night. In accordance, dry weight and class number of the hepatocytes were both higher by day than by night. 3. By reversal of the light-dark cycle, circadian rhythms of liver weight, hepatocyte mitotic activity and serum corticosterone underwent a reversal. In accordance, circadian rhythm also reversed of both dry mass of the hepatocytes, which became heavier by night than by day, and pattern of the hepatocyte weight-classes, which became sharper, more discrete and more numerous by night, less defined and lower in number by day. 4. Feeding restriction to early morning or to late afternoon did not affect substantially the circadian rhythms of the parameters examined. 5. Binuclear cell frequency did never differ significantly at midnight with respect to midday, irrespectively to the experimental condition. 6. Regulation of the circadian rhythm of both weight-class pattern and dry mass of the hepatocytes appears to be mainly acted by the light-dark regimen likely via modulation of the plasma glucocorticoids (corticosterone) concentration, and increase/decrease of which causes a decrease/increase of the total solid content of hepatocytes, with redistribution of cells in the weight-classes. 7. Feeding rhythm and time elapsed from food intake mainly influence definition of the individual weight-classes and weight range of the hepatocytes

Metyrapone modulation of tyrosine aminotransferase induction by dexamethasone in cultured hepatocytes.

Metyrapone, an inhibitor of cytochrome P-450-dependent monooxygenases, enhanced the induction of tyrosine aminotransferase by dexamethasone in primary cultures of hepatocytes, while it had no effect on the basal level of the enzyme activity in the absence of the hormone. The amplification of the hormonal induction of tyrosine aminotransferase activity was strictly correlated with the concentration and with the inhibitory action of the compound on cytochrome P-450. The phenomenon occurred even at the maximally effective concentrations of dexamethasone, thus showing that metyrapone is a 'Glucocorticoid Potency Amplifier'. The dexamethasone activity amplification by metyrapone could be the consequence of a modulation of the glucocorticoid biotransformations due to the cytochrome P-450 inhibitor

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

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