Improvement in liver fibrosis, functionality and hemodynamics in CCI4-cirrhotic rats after injection of the Liver Growth Factor.

BACKGROUND/AIMS: Most substances used in experimental models of cirrhosis are chosen either as protectors of lipid peroxidation, as antifibrogenic agents or as vitamins, among others. In this report, we analyze the improvement produced, in established cirrhosis (CCl4 plus phenobarbital) in rats, by intraperitoneal injection of Liver Growth Factor, a hepatic mitogen with activity both in vivo and in vitro. METHODS: Following confirmation of CCl4-induced cirrhosis, Liver Growth Factor (4.5 microg per ratx2 injections/week for 3 weeks) was administered to one group of rats (Cirr+LGF). The remaining rats (Cirr) received saline. The groups were compared in terms of serum enzymes, tissue damage, total liver collagen, collagenase activity, microsomal enzyme activities, splanchnic and systemic hemodynamics and portosystemic shunting. RESULTS: Treatment of rats presenting CCl4-induced cirrhosis with Liver Growth Factor decreased serum aminotransferase levels and increased levels of serum albumin and total protein. The Liver collagen content was lower in rats treated with Liver Growth Factor (2.96 vs. 4.32 mg/g liver, p<0.01). Microscopic studies revealed that the livers of rats receiving Liver Growth Factor showed decreases in fibrosis, necrosis and inflammatory infiltration, as well as a recovery of architectural integrity. Liver function was improved after treatment with Liver Growth Factor, as indicated by the rate constant for elimination of aminopyrine, which increased from 0.0063 to 0.0170 (p<0.05). This increase was accompanied by a higher total amount of cytochrome P-450 as well as of certain P-450 isoenzymes, especially those that are hormone-dependent, such as P-450 3A. The improved liver histology and function observed in Cirr+LGF rats was associated with decreases in portal pressure (14.4 vs. 9.4 mm Hg, p<0.01) and portosystemic shunting (55.8 vs. 11.5%, p<0.01), as well as increases in mean arterial pressure and systemic vascular resistance, and a reduction in ascites. CONCLUSIONS: Administration of the hepatic mitogen, Liver Growth Factor, to CCl4-cirrhotic rats decreased liver collagen and reorganized the hepatic extracellular matrix, resulting in an improvement in liver function, reduced portal pressure and amelioration of ascites.

Na+ :H+ exchange inhibition induces intracellular acidosis and differentially impairs cell growth and viability of human and rat hepatocarcinoma cells.

Amiloride and its more potent analog, hexamethylene amiloride (HMA), inhibits Na+ :H+ exchange and decreases intracellular pH in a concentration-dependent way in two human hepatocarcinoma cell lines and in a rat hepatocarcinoma cell line that differs in its phenotypic characteristics, resembling the clinical situation encountered in human hepatocarcinomas. After 24 h of exposure, DNA synthesis and cell protein content of the cultures decreases according to the concentration of the drugs and in parallel to Na+ exchange inhibition and the drop in pHi promoted. RNA and protein syntheses are less sensitive to its action. The above effects induced by HMA are accompanied by an abrupt decrease in cell viability and lysosomal integrity at 24 h. These effects develop gradually with the exposure time as does the increase in free radical production. Decreased viability is totally or partially restored by N-acetylcysteine or deferoxamine, but the degree of intracellular acidification produced is not. These results tend to suggest that intracellular acidification can diminish cell growth and provoke cytotoxic cell death by diminishing reduced glutathione (GSH) levels and impairing lysosomal integrity, reflecting the sensitivity of hepatocarcinoma cells to Na+ exchange inhibition and intracellular acidosis.

Attenuation of apoptotic DNA fragmentation by amiloride.

Amiloride is a K+-sparing diuretic that effectively inhibits the Na+/H+ transporter in the plasma membrane of most mammalian cells. We have examined the effects of amiloride on the progression of apoptosis in HL-60 cells induced by camptothecin (CAM), cycloheximide (CHX), and 20 Gy gamma irradiation. Spectrofluorometric measurements on cell populations showed an inhibition of Na+/H+ transporter activity and a corresponding decrease in intracellular pH following treatment with amiloride alone, or in combination with the apoptosis-inducing agents. Flow cytometric cell cycle analysis, in combination with DNA strand break analysis, indicated that amiloride diminished endonuclease-mediated degradation of nuclear chromatin 3 h following treatment with CAM or CHX, and prevented degradation for 3 h following gamma radiation treatment. Apoptosis-associated DNA degradation was significantly greater for all three agents in the absence of amiloride. Protection from radiation-induced apoptosis was transient, since apoptotic subpopulations were observed, but still at a decreased level, 5 h following irradiation. Amiloride was as effective as zinc, an inhibitor of Ca2+/Mg2+-dependent endonucleases, in reducing or delaying the onset of endonuclease activity. Data presented show that effects of amiloride on membrane Na+/H+ transporter activity and intracellular pH can potentially affect apoptotic signaling cascades, leading to a retardation in the rate of progression to an apoptotic cell death. Results also point to the involvement of intracellular pH and Ca2+ in the regulation of apoptotic endonuclease activity, and the need for a functional Na+/H+ exchanger for the induction of apoptosis.

Hydrogen ion-dependent oncogenesis and parallel new avenues to cancer prevention and treatment using a H(+)-mediated unifying approach: pH-related and pH-unrelated mechanisms.

A comprehensive examination of phenomena related to cancer is presented that is based on hydrogen ion dynamics, as viewed from the biological, biochemical, and biophysical perspective. A model is described that considers an array of cancer-associated events from oncogenesis to carcinogenesis from this perspective. The basic ideas are viewed from various aspects, ranging from the cellular level to the clinical situation. The novel types of therapeutic and prophylactic agents that result from applying these concepts are elucidated. Considerable insight into this modern approach is seen from some of the mechanisms that characterize the phenomenon of spontaneous regression of cancer.

Hepatic growth induced by injection of the liver growth factor into normal rats.

Normal Wistar rats injected with the liver growth factor (LGF), a mitogen specific for liver cells, experienced hepatic growth. LGF shows two peaks of activity in vivo, both of them mitogenic. Rats injected either with 6.8 ng or 3.9 micrograms LGF/rat every 3-4 days experienced liver growth showing a see-saw profile. Dry liver weight usually peaked at day 2 (microgram doses) or at day 3 (ng doses) after each injection, with increases of about 30% over controls. Liver DNA synthesis, measured by [3H]-thymidine incorporation, peaked 24 h after LGF injection at both doses. Liver protein synthesis, measured by [14]C-leucine incorporation, usually peaked 24 h after DNA synthesis maximums. Mitogen-stimulated cells were also assessed by immunohistochemical staining for proliferating cell nuclear antigen in livers of LGF-injected rats. Rats injected with rat serum albumin purified from normal rats to serve as controls showed a 6% increase in dry liver weight, but when serum albumin from 3-day fasted rats was injected instead, the increase was not statistically significant. The mild effect of rat serum albumin could be due to the lipid content of the solutions injected, but the level of lipids/mg protein in LGF solutions was half that determined with serum albumin from 3-day fasted rats. From the microscopic and ultramicroscopic studies carried out in rat livers injected with LGF at each dose, we observed: (1) an increase in the number of hepatocytes undergoing mitosis; (2) transient increases in lipid and glycogen contents, as occur after liver resection; (3) no signs of degeneration, such as the appearance of amyloids or fibrosis; (4) no increase in lysosome number, as in hepatotoxicity; (5) no alterations in endothelial or Kupffer cells; and (6) no ultrastructural signs of degeneration either in cytoplasmic organelles (rough endoplasmic reticulum, mitochondria) or in nuclei. One year after LGF injection, rat liver, pancreas, kidneys and spleen were normal, with no signs of degeneration or onset of fibrosis.

Biological properties of a renotropic protein present in plasma of uninephrectomized rats.

The biological characteristics of a kidney growth factor (KGF) from uninephrectomized rat plasma have been studied. A crude preparation of this factor [Nephrol. Dial. Transplant. 4: 334-338, 1989] was further purified by hydrophobic interaction HPLC and gel filtration. KGF was found to be a heat- and trypsin-resistant protein. This factor stimulated dose-dependently DNA synthesis by the mouse kidney in vivo, and by either rat renal tubules or serum-deprived LLC-PK1 cells, in vitro. KGF also increased protein synthesis in these cells, in a dose-dependent manner. Moreover, KGF stimulated sodium uptake by these cells, associated with the maximal increase of protein synthesis. Our findings indicate that KGF is a potent renotropic protein which can play a key role in the renal compensatory growth after uninephrectomy.

Co-purification of calcium transport-stimulating and DNA synthesis-stimulating agents with parathormone-like activity isolated from the hypercalcaemic strain of the Walker 256 tumour.

One of the strains of the Walker 256 carcinosarcoma induces in the rat a humoral hypercalcaemia of malignancy (HHM) syndrome which is similar to that reported in human patients. We have isolated from this tumour a chromatographic fraction which displays an adenylate cyclase stimulating activity in dog kidney cortical membranes, similar to that of a parathormone (PTH) related protein isolated from various HHM related tumours. In addition, this fraction stimulated initial calcium (Ca) uptake in confluent Madin-Darby canine kidney (MDCK) cell monolayers in a dose-dependent manner. Maximal stimulation of Ca uptake was associated with an enhanced Ca efflux from MDCK cells preloaded with the cation, and with an increased DNA synthesis in these cells. These activities might be involved in development of increased tubular calcium reabsorption in Walker 256 tumour-bearing rats.

Liver growth factor purified from human plasma is an albumin-bilirubin complex.

We have reported that a liver growth factor isolated from plasma of partially hepatectomized rats is an albumin-bilirubin complex. In this paper, we characterize the liver growth factor purified from subjects with hepatitis (h-LGF). This factor increases synthesis of DNA in a dose-dependent manner both in vivo in mouse hepatocytes, with a dose of maximal stimulation of 150 ng of h-LGF/mouse, and in vitro in rat liver cell culture, with maximal effect at 7.5 to 10 ng of h-LGF/ml. In vivo, h-LGF increases the mitotic index of mouse hepatocytes, its action being organ-specific, acting on liver, but not on spleen, kidney, lung or brain. In vitro, h-LGF stimulates the uptake of 22Na+ by hepatocytes. In addition, we carried out a study comparing it with human serum albumin in terms of absorbance, fluorescence, circular dichroism spectra, amino acid composition, tryptic maps and antigenic determinants (Ouchterlony immunodiffusion). All these tests suggested that human serum albumin is a constituent of h-LGF. Moreover, when albumin isolated from humans without hepatic pathology is incubated with bilirubin, the albumin-bilirubin complex formed mimics the activity of the human liver growth factor with respect to stimulation of DNA synthesis and the effects on the mitotic index of mouse hepatocytes in vivo. We propose that this human liver growth factor is an albumin-bilirubin complex.

Partial purification and characterisation of a renal growth factor from plasma of uninephrectomised rats.

Renal growth factor activity was extracted from plasma of adult uninephrectomised rats and partially purified by gel filtration and anion-exchanger FPLC. It induced a maximal stimulation of mouse DNA synthesis in vivo at 1.75 micrograms/mouse. In addition, renal growth factor was found to maximally stimulate DNA synthesis in LLC-PK1 cells at 150 ng/ml. This maximal response was then found to decrease with higher doses of renal growth factor, in vivo and in vitro. The apparent molecular weight of renal growth factor was estimated to be 17K-22 K by gel filtration. It was found to be resistant to heat and to trypsin, but labile to reduction with dithiothreitol.

Effect of L-thioproline on the progression and events of the cell cycle of HeLa cells.

L-thioproline (thiazolidine-4-carboxylic acid, L-Tp) reduces DNA synthesis in cultured HeLa cells by about 50% when added to synchronized cultures at the beginning of G1 phase. This inhibition is not observed when the drug is added at mid-S, in G2 or in M. L-Tp almost annuls the burst in 22Na uptake occurring at the beginning of G1 and is as potent an inhibitor as amiloride. It is also able to delay the apparition of the early S-specific cAMP peak and to elevate its intracellular concentration. These results indicate that L-Tp could decrease tumor cell growth by acting at the G1-S boundary.

Diferric transferrin reduction stimulates the Na+/H+ antiport of HeLa cells.

Proton release from HeLa cells is stimulated by external oxidants for the transplasmalemma electron transport enzymes. These oxidants, such as ferricyanide and diferric transferrin, also stimulate cell growth. We now present evidence that proton release associated with the reduction of ferricyanide and diferric transferrin is through the Na+/H+ antiport. The stoichiometry of H+/e- release with diferric transferrin is over 50 to 1, which is greater than expected for oxidation of a protonated transmembrane electron carrier. Diferric transferrin induced proton release depends on external sodium and is inhibited by amiloride. Proton release is also inhibited when diferric transferrin reduction is inhibited by apotransferrin. A tightly coupled association between the redox system and the antiport is shown by sodium dependence and amiloride inhibition of diferric transferrin reduction. The results indicate a new role for ferric transferrin in growth stimulation by activation of the sodium-proton antiport.

Identification of a liver growth factor as an albumin-bilirubin complex.

We have reported the purification and characterization of a protein that behaves as a liver growth factor, showing activity either in vivo or in vitro [Díaz-Gil et al. (1986) Biochem. J. 235, 49-55]. In the present paper, we identify this liver growth factor (LGF) as an albumin-bilirubin complex. This conclusion is supported by the results of chemical and spectroscopic characterization of this protein as well as by experiments in vivo. Incubation of albumin isolated from normal rats with bilirubin/albumin molar ratios (r) resulted (when r = 1 or 2) in a complex with liver DNA synthesis promoter activity identical with that of LGF. The exact amount of bilirubin bound to albumin was assessed by fluorescence and c.d. spectra. This albumin-bilirubin complex showed the same dose-dependence profile as LGF either at low or high dose of protein injected per mouse. Both LGF and albumin-bilirubin complex produced similar increases in the mitotic index of mouse hepatocytes in vivo. A new mechanism for the onset of the hepatic regenerative process is proposed.

Purification of a liver DNA-synthesis promoter from plasma of partially hepatectomized rats.

A protein was isolated from plasma of partially (70%) hepatectomized rats that, injected in mice, increases the uptake of [3H]thymidine by liver DNA by 200-300% over that by injected control saline. The purification procedure consists essentially of three chromatography steps, employing Sephadex G-75, DEAE-cellulose and hydroxyapatite. The hepatic promoter (HP) preparation shows a single band in SDS/polyacrylamide (15%)-gel electrophoresis (silver stained), with an Mr of 64 000; its activity is suppressed by trypsin or pepsin and is unaffected by deoxyribonuclease or ribonuclease. On injection into mice (150 ng/mouse), it increases the mitotic index of the liver. It shows organ-specificity, acting on liver but not on spleen, kidney, lung or brain. In primary liver cultures, it produces an increase in uptake of [3H]thymidine into DNA in the range 1-10 ng/ml. In this system in vitro, it increases the uptake of 22Na+ immediately after addition.

Surprising differences in substrate selectivity and other properties of systems A and ASC between rat hepatocytes and the hepatoma cell line HTC.

We find that the two wide-range Na+-dependent transport systems A and ASC for various neutral amino acid can be discriminated more sharply in the hepatoma cell line HTC than in any cell yet studied by us in which the two systems co-exist. The gain comes partly from a higher reproducibility and a higher relative ASC rate for HTC than in ordinary rat hepatocytes, also a repressed condition of System A unless first deprived of amino acids, but mainly from our finding that in the hepatoma cell threonine serves as a nearly specific substrate and inhibitor of System ASC, thus decisively supplementing older discriminatory techniques. In ordinary hepatocytes cysteine is quite specific to ASC as a substrate but not as an inhibitor, whereas threonine is specific in neither role. In the hepatoma cell cysteine in turn is specific in neither role. In addition to these and other differences between the two cells in analog specificity, which are partly assignable to System ASC and partly to System A, System ASC of the hepatoma cell shows an inhibition on lowering the pH from 6.5 to 5 not seen in the ordinary hepatocyte. Furthermore, threonine uptake by the hepatoma cell undergoes no stimulation when Li+ is substituted for choline in a Na+-free medium, whereas ASC uptake by the ordinary rat hepatocyte is stimulated much as is System A uptake. As in other occurrences, and in contrast to System A, ASC transport in the hepatoma cell is stimulated neither by amino acid deprivation nor by insulin, glucagon, or dexamethasone. Trans-stimulation, both inward and outward, via System ASC is vigorous in the hepatoma cell. Despite the surprising differences observed, common features of each system in various occurrences continue to justify the use of the abbreviations ASC and A as long as they are understood as generic designations.

Measurement of enzymatic activities in turbid suspensions by double beam spectrophotometry.

A method for the determination of enzyme activities in turbid suspension is described, based on the use of a double beam or double wavelength spectrophotometer in one or two cell mode. It measures the disappearance of NADH, coupled to the activity of the enzyme, at a 340 wavelength or at a 340 minus 374 mmu wavelength pair. The method has been applied to the determination of pyruvate kinase activity, in the presence of isolated mitochondria, and other possible applications are discussed.

Action of the antipsychotic drugs thiotixene and thioridazine on isolated mitochondria.

Thiotixene and thioridazine antipsychotic drugs produce a stimulation of mitochondrial respiration in state 3 and state 4 resulting in a complete respiratory control loss, only accompanied by slight alterations of oxidative phosphorylation. This effect may be of biological significance in the action mechanism of the drugs.

Effects and specificity of anticancer agents on the respiration and energy metabolism of tumor cells.

Using 35 new anticancer agents from the screening program of the National Cancer Institute we have performed a biochemical study of the effects on the respiration and oxidative phosphorylation of rat liver mitochondria and on the respiration of leukocyte, liver, L1210 leukemia, and Ehrlich ascites cell suspensions. Fifteen of the 35 compounds were found to be potent respiratory inhibitors as defined by 50% inhibition of mitochondrial respiration at concentrations of 110 mu mol/liter or less. The mechanism of respiratory inhibition by the drugs was either a rotenone-, antimycin-, or oligomycin-like effect. One triazine derivative showed some specificity for inhibiting tumor cell respiration in comparison with normal cell respiration. Two naphthoquinone derivatives showed inhibition of respiration in in vivo treatments at chemotherapeutic doses. It was concluded that data on respiratory effects may assist in the interpretation of the results of in vivo and in vitro screening tests of the drugs, and that in some cases, as with the naphthoquinone derivatives, the effects on respiration could be related to the mechanism of action or the mechanism of toxicity of the drugs.