I. Quantitative determination of the amount of DNA per nucleus by interference microscopy.

A method for the determination of the DNA content of isolated nuclei of different ploidy has been developed. It is based on measurement of the nuclear dry mass, with an integrating microinterferometer, before and after DNase treatment. The values found are slightly low, because, as indicated by biochemical determinations, consistently 5% to 8% of DNA is not extracted by DNase under these conditions. The average DNA values thus obtained for diploid and tetraploid nuclei of adult rat liver are 7.7 and 15.6 pg (10(-12) g), respectively. Definite advantages of this procedure are: i) comparisons with biochemical determinations to give DNA values for each class of ploidy, ii) comparisons with histophotometry of the Feulgen dye-DNA complex to give absolute values instead of arbitrary units.

II. Differences in adrenal medulla nuclear DNA content among rats of different strains following intermittent exposure to cold.

The amount of DNA per nucleus in the adrenal medulla cells of four different strains of rats (Wistar, Sprague-Dawley, Long-Evans, and Italico) is determined both under control conditions and after 300 hr of intermittent exposure to cold. The adrenal medulla nuclei of the four strains of rats contain the same amount of DNA; however, the loss of DNA observed after the same experimental treatment differs markedly in the different strains. The loss is small in Wistar and Sprague-Dawley rats (8-13%), larger in Long-Evans rats (20%) and still larger in Italico rats (45%). The DNA loss in Wistar rats increases if the animals are fed the same diet as the Italico rats, and the DNA loss in Italico rats is reduced if the animals are fed the same diet as the Wistar rats. The different behavior of the four strains is discussed in terms of turnover of DNA.

gamma-Glutamyl transpeptidase-dependent lipid peroxidation in isolated hepatocytes and HepG2 hepatoma cells.

Gamma-glutamyltranspeptidase (GGT), a plasma membrane-bound enzyme, provides the only activity capable to effect the hydrolysis of extracellular glutathione (GSH), thus favoring the cellular utilization of its constituent amino acids. Recent studies have shown however that in the presence of chelated iron prooxidant species can be originated during GGT-mediated metabolism of GSH, and that a process of lipid peroxidation can be started eventually in suitable lipid substrates. The present study was undertaken to verify if a GGT-dependent lipid peroxidation process can be induced in the lipids of biological membranes, including living cells, and if this effect can be sustained by the GGT highly expressed at the surface of HepG2 human hepatoma cells. In rat liver microsomes (chosen as model membrane lipid substrate) exposed to GSH and ADP-chelated iron, the addition of GGT caused a marked stimulation of lipid peroxidation, which was further enhanced by the addition of the GGT co-substrate glycyl-glycine. The same was observed in primary cultures of isolated rat hepatocytes, where the lipid peroxidation process did not induce acute toxic effects. GGT-stimulation of lipid peroxidation was dependent both on the concentration of GSH and of ADP-chelated iron. In GGT-rich HepG2 human hepatoma cells, the exposure to GSH, glycyl-glycine, and ADP-chelated iron resulted in a nontoxic lipid peroxidation process, which could be prevented by means of GGT inhibitors such as acivicin and the serine-boric acid complex. In addition, by co-incubation of HepG2 cells with rat liver microsomes, it was observed that the GGT owned by HepG2 cells can act extracellularly, as a stimulant on the GSH- and iron-dependent lipid peroxidation of microsomes. The data reported indicate that the lipid peroxidation of liver microsomes and of living cells can be stimulated by the GGT-mediated metabolism of GSH. Due to the well established interactions of lipid peroxidation products with cell proliferation, the phenomenon may bear particular significance in the carcinogenic process, where a relationship between the expression of GGT and tumor progression has been envisaged.

Redox modulation of cell surface protein thiols in U937 lymphoma cells: the role of gamma-glutamyl transpeptidase-dependent H2O2 production and S-thiolation.

The expression of gamma-glutamyl transpeptidase (GGT), a plasma membrane ectoenzyme involved in the metabolism of extracellular reduced glutathione (GSH), is a marker of neoplastic progression in several experimental models, and occurs in a number of human malignant neoplasms and their metastases. Because it favors the supply of precursors for the synthesis of GSH, GGT expression has been interpreted as a member in cellular antioxidant defense systems. However, thiol metabolites generated at the cell surface during GGT activity can induce prooxidant reactions, leading to production of free radical oxidant species. The present study was designed to characterize the prooxidant reactions occurring during GGT ectoactivity, and their possible effects on the thiol redox status of proteins of the cell surface. Results indicate that: (i) in U937 cells, expressing significant amounts of membrane-bound GGT, GGT-mediated metabolism of GSH is coupled with the extracellular production of hydrogen peroxide; (ii) GGT activity also results in decreased levels of protein thiols at the cell surface; (iii) GGT-dependent decrease in protein thiols is due to sulfhydryl oxidation and protein S-thiolation reactions; and (iv) GGT irreversible inhibition by acivicin is sufficient to produce an increase of protein thiols at the cell surface. Membrane receptors and transcription factors have been shown to possess critical thiols involved in the transduction of proliferative signals. Furthermore, it was suggested that S-thiolation of cellular proteins may represent a mechanism for protection of vulnerable thiols against irreversible damage by prooxidant agents. Thus, the findings reported here provide additional explanations for the envisaged role played by membrane-bound GGT activity in the proliferative attitude of malignant cells and their resistance to prooxidant drugs and radiation therapy.

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.

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.

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.

Gamma-glutamyltranspeptidase activity in human ovarian carcinoma.

Gamma-glutamyltranspeptidase (GGT) is a cell membrane enzyme involved in the hydrolysis and uptake of extracellular glutathione. Histochemically detectable GGT has been shown in several human neoplasms. However, few studies have addressed the quantitative biochemical assessment of GGT activity in human tumors, and the importance of GGT activity in human tumor biology remains to be elucidated. The aim of the present study was to assess biochemically GGT enzyme activity in human ovarian surgical biopsies. GGT activity was assayed in homogenates of surgical samples of ovarian tumors and compared with the clinical data of the patients in order to establish: a) the level of tumor GGT activity, b) its correlation with other clinical parameters of the neoplasms, c) the possibility of the induction in vivo of GGT after anticancer platinum-based therapy, since some of the patients were pretreated. The results indicated that ovarian tumor expresses biochemically relevant GGT activity. The sensitive method used in this study allowed the quantitative evaluation of enzyme activity in all samples examined, showing that GGT activity values in ovarian carcinoma samples were extremely variable among the different subjects, both in untreated neoplasms (6.2 +/- 5.4 mU/mg protein) and in second-look laparotomy biopsies following platinum-based therapy (4.7 +/- 3.8 mU/mg protein). The mean GGT activity in benign ovarian tumors was lower than that in malignant tumors. No significant correlation was found between GGT activity and patient characteristics (tumor stage, age of patients, serum CA125, TAG-72, and GGT levels). However, the biological and pharmacological relevance of GGT expression remain to be elucidated in a large series of tumors.

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.

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.

Gamma-glutamyl transpeptidase-dependent iron reduction and LDL oxidation--a potential mechanism in atherosclerosis.

BACKGROUND: gamma-Glutamyl transpeptidase (gamma-GT) is found in serum and in the plasma membranes of virtually all cell types. Its physiologic role is to initiate the hydrolysis of extracellular glutathione (GSH), a tripeptide in which cysteine lies between alpha-glycine and gamma-glutamate residues. Cysteine and other thiol compounds are known to promote LDL oxidation by reducing Fe(III) to redox active Fe(II); therefore, we sought to determine whether similar reactions can be sustained by GSH and influenced by gamma-GT. METHODS: Fe(III) reduction and LDL oxidation were studied by monitoring the formation bathophenanthroline-chelatable Fe(II) and the accumulation of thiobarbituric acid-reactive substances, respectively. Human atheromatous tissues were examined by histochemical techniques for the presence of oxidized LDL and their colocalization with cells expressing gamma-GT activity. RESULTS: A series of experiments showed that the gamma-glutamate residue of GSH affected interactions of the juxtaposed cysteine thiol with iron, precluding Fe(III) reduction and hence LDL oxidation. Both processes increased remarkably after addition of purified gamma-GT, which acts by removing the gamma-glutamate residue. GSH-dependent LDL oxidation was similarly promoted by gamma-GT associated with the plasma membrane of human monoblastoid cells, and this process required iron traces that can be found in advanced or late stage atheromas. Collectively, these findings suggested a possible role for gamma-GT in the cellular processes of LDL oxidation and atherogenesis. Histochemical analyses confirmed that this may be the case, showing that gamma-GT activity is expressed by macrophage-derived foam cells within human atheromas, and that these cells colocalize with oxidized LDL. CONCLUSIONS: Biochemical and histochemical correlates indicate that gamma-GT can promote LDL oxidation by hydrolyzing GSH into more potent iron reductants. These findings may provide mechanistic clues to the epidemiologic evidence for a possible correlation between persistent elevation of gamma-GT and the risk of fatal reinfarction in patients with ischemic heart disease.

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.

Dry weight, triglycerides content and glucose-6-phosphatase activity of hepatocytes separated by sedimentation.

1) Isolated rat-hepatocytes were subfractionated by isopycnic or velocity sedimentation, and dry mass, triglycerides content and Glucose-6-Phosphatase activity of different subpopulations were determined. 2) Distribution of the cell dry masses and dry mass per average cell were substantially similar in all the fractions separated by isopycnic sedimentation. Velocity sedimentation allowed a satisfactory separation of cells of different dry mass. 3) Triglycerides content and Glucose-6-Phosphatase activity of cells of the different fractions obtained by isopycnic sedimentation showed no statistically significant difference. Subpopulations fractionated by velocity sedimentation differed in both triglycerides content and Glucose-6-Phosphatase activity, which were substantially parallel to the cell dry mass.

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.

A quantitative cytochemical study of isolated epithelial cells of the hamster cheek pouch.

1. Dissociation of the hamster cheek pouch epithelium by a method combining trypsin attack and EDTA exposure, yielded a mixture f highly viable isolated cells (basal, spinous, and granular cells). 2. Such heterogeneous cell population was reproducibly separated into 4 fractions by pycnic sedimentation: in order of increasing density, Fraction I and Fraction II predominantly contained cells of the basal layer (93% and 76%, respectively), Fraction III basal, spinous and granular cells in equivalent concentration, Fraction IV mainly granular cells (69%). Horny squamae sedimented at the bottom of the tube. 3. Individual cells of the whole population and those of the population fractions separated by density gradient, were analyzed and charcterized as regards morphology, viability, volume, dry mass, density and DNA synthesizing ability. 4. A positive correlation was found between morphology, dry mass and density of the cells, showing that differentiation occurs by increments in mass and density. The weights of the basal, spinous and granular cells were distributed within ranges well defined and scantily overlapping, and were positively correlated with cell differentiation. Also cell density increased with differentiation degree. On the contrary, volume distribution of the various types of cells showed differences of minor importance, indicating for an increase of solids concentration in the more differentiated cells. 5. DNA synthesis took place only in basal cells, the density of which was generally very low.

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.

Microinterferometric characterization of isolated human hepatocytes.

Hepatocytes isolated from liver tissue taken by biopsy from 18 patients with hepatic or extrahepatic diseases displayed a weight class-organization similar to that of other animal species. In most cases the cell classes had a period of 108 pg varying in number from 4 to 14; cell weight range was 96--432 pg as a minimum (4 classes) and 108--1536 pg as a maximum (14 classes). In 5 cases cell classes showed a period of 120 pg resulting 7--9 in number; cell weight range was 216--960 pg (7 classes) or 216--1200 pg (9 classes). No correlation was found between sex, age, liver histopathology, disease of the patients and the various parameters measured.

Changes in protein sulfur groups in hepatocytes of newborn rats under glucocorticoid stimulation.

A daily administration of hydrocortisone-acetate (25 micrograms/g b.w.) increased both dry mass and protein content of the hepatocytes of newborn rats by 84% and 89% respectively, after 5-day-treatment. The increase correlated with the duration of hormonal treatment. As an average per cell, the total reactive protein sulfur increased up to 127%. This increase depends on a major increment of thiols (+179%) and on a minor increment of disulfides (+18%). Within the thiols, the fast reactive ones exhibited the most pronounced increment (+214%). Per protein unit, the total reactive sulfur increased, after a 1 day lag period, by up to +20%. Thiols showed a 47%-increment due to increase of fast reactive thiols (+70%) more than of slow reactive thiols (+20%). On the contrary, disulfides decreased (-37%). Consequently, the protein thiol/disulfide ratio shifted from 2.14 in control hepatocytes to 4.98 (+133%) in hormone-stimulated hepatocytes. Both the increase of the thiol content, and the shift of the SH/SS-equilibrium of the cellular proteins, correlated with a concomitant increase of enzymic activities such as gamma-glutamyltranspeptidase, glutathione reductase, glutathione peroxidase, glutathione S-transferase and 7-ethoxycoumarin O-deethylase.