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Much has been learned in the past few years concerning the morphology and function of the intrahepatic biliary epithelium. Immunohistochemistry, together with ultrastructural studies has allowed a better identification or the smallest branches of the biliary tree and of subcellular components (cytoskeleton, specializations of the cell membrane, specific receptors). Modulation of the biliary epithelium in response to physiological or pathological stimuli has renewed the interest concerning the existence of facultative stem cells in the liver. More information is needed however concerning the mechanisms of cell loss in vanishing bile duct syndromes.

Background/aims: The involvement of a direct viral cytopathic effect or an immune-mediated mechanism in the progression of hepatic damage in chronic hepatitis C is controversial. The type of immune response is itself a matter of controversy, and histological data are lacking. The aim of this study was to identify the factors associated with the progression of liver injury in 30 HCV/RNA-positive untreated patients with chronic hepatitis., Methods: Necroinflammatory and architectural damage were evaluated using Ishak's score. Activated hepatic stellate cells (HSC) were visualized by immunohistochemistry for alpha-smooth muscle actin (alphaSMA) and quantitated by morphometry. Plasma HCV/RNA was evaluated using a competitive RT-PCR method. To study the type of immune response involved in the progression of liver injury, interferon gamma (IFNgamma)-positive cells (as expression of a Th1-like response) were evaluated by immunohistochemistry and quantitated by morphometry., Results: HSC were mostly detected close to areas of lobular necroinflammation or lining fibrotic septa. The alphaSMA- and Sirius Red-positive parenchyma correlated significantly with necroinflammatory and architectural scores. IFNgamma-positive cells were detected in periportal areas associated with the inflammatory infiltrates and significantly correlated with architectural damage. No relationship was found between the histological features of liver injury and viral load., Conclusions: HSC activation and progression of liver injury are unrelated to viral load but associated with a Th1-like response, a plausible target for the treatment of chronic hepatitis C.

Background & Aims: The Na+/H+ exchanger is the main intracellular pH regulator in hepatic stellate cells (HSCs), and its activity is increased by platelet-derived growth factor (PDGF). Amiloride, an Na+/H+ exchange inhibitor, reduces PDGF-induced HSC proliferation, suggesting that the Na+/H+ exchanger plays a role in regulating HSC proliferative response. The aim of this study was to characterize the intracellular pathways mediating activation of the Na+/H+ exchanger by PDGF in HSCs., Methods: The activity of the Na+/H+ exchanger and HSC proliferation rate were evaluated under control condition and after incubation with PDGF in the absence or presence of specific inhibitors of the main intracellular pathways of signal transduction. Na+/H+ exchange protein expression was evaluated by means of Western blot., Results: PDGF induced a significant increase in the activity of the Na+/H+ exchanger without modifying protein expression. Inhibition of the calcium/calmodulin- and protein kinase C-dependent pathways resulted in a significant inhibition of both Na+/H+ exchange activity and of PDGF-induced HSC proliferation. The involvement of the two pathways was confirmed by showing that incubation of HSCs with both phorbol-12-myristate-13-acetate, a potent protein kinase C activator, and thapsigargin, which increases intracellular calcium levels, significantly increased both the Na+/H+ exchanger activity and HSC proliferation rate. Inhibition of the protein kinase A pathway did not modify either PDGF-induced Na+/H+ exchange activation or PDGF-induced HSC proliferation. On the contrary, inhibition of the mitogen-activated protein kinase- and of phosphatidylinositol 3-kinase-dependent pathways significantly reduced PDGF-induced HSC proliferation without affecting the activity of the Na+/H+ exchanger., Conclusions: Activation of the Na+/H+ exchanger by PDGF in HSCs is mediated by calcium/calmodulin- and protein kinase C-dependent pathways. PDGF-induced HSC proliferation is mediated by Na+/H+ exchange-dependent and -independent pathways.

Background/aims: Oxidative stress is associated with liver fibrosis in vivo and with hepatic stellate cell (HSC) activation in vitro, but the intracellular mechanisms mediating these effects are mostly unknown. The Na+/H+ exchanger plays a key role in regulating the cell cycle, and is involved in HSC proliferation. Its role in different HSC features, such as collagen accumulation, is still unknown. We thus evaluated if the Na+/H+ exchanger modulates the fibrogenic effect of oxidative stress in rat HSC., Methods: HSC were incubated with 0.1 mM ferric nitrilotriacetate complex (FeNTA). Intracellular hydroperoxides and malonildialdehyde (MDA) levels in the culture media were measured by the dichlorofluorescein and TBARS method, respectively. Intracellular pH and Na+/H+ exchanger activity were measured using the fluorescent dye BCECF. Cell proliferation was measured by immunohistochemistry for bromodeoxyuridine incorporation. Collagen type I accumulation in the culture media was measured by ELISA., Results: HSC incubation with FeNTA resulted in a significant production of intracellular hydroperoxides and MDA, associated with increased Na+/H+ exchange activity and baseline intracellular pH (pHi). Exposure of HSC to FeNTA significantly enhanced the number of proliferating HSC and collagen type I levels in the culture medium. All these effects were reversed by the antioxidant resveratrol and by the Na+/H+ exchanger inhibitor amiloride., Conclusions: This study indicates that the Na+/H+ exchanger might represent a common mediator of the different effects induced by oxidative stress on HSC. The reduction in cell proliferation and collagen synthesis induced by amiloride could represent a new therapeutic challenge in liver fibrosis.

The present study was conducted to evaluate if the increased rate of apoptosis previously reported in the liver of ethanol-treated rats was accompanied by increased cell renewal. A quantitative analysis of apoptosis was performed in rats fed an ethanol-containing liquid diet for 5 weeks. S-phase cells were demonstrated by immunohistochemistry, using the Bromodeoxyuridine/anti-Bromodeoxyuridine method. In ethanol-fed rats apoptosis was five times greater than in pair-fed controls. Bromodeoxyuridine-labelled hepatocytes increased from 0.07 +/- 0.009% in controls to 0.17 +/- 0.013% (p < 0.001) and Bromodeoxyuridine-labelled lipocytes (desmin-positive sinusoidal cells) increased from 3.43 +/- 0.28% to 6.60 +/- 1.04% (p < 0.001). The lobular distribution of labelled cells was modified with a shift towards the perivenular areas. The results of this study suggest that the replacement of liver cells lost by ethanol-induced apoptosis is not impaired in intact (non-operated) animals. The impaired regeneration following partial hepatectomy reported in ethanol-fed rats is possibly due to differences in the extent of parenchymal loss, to altered relationships between hepatocytes and blood supply and to the modalities of regeneration involved.

The expression of proliferating cell nuclear antigen (PCNA) was examined in normal human and rat liver fixed in either formaldehyde or methanol, and was compared with the incorporation of bromodeoxyuridine (BrdU) in S-phase cells. Codistribution of PCNA and BrdU was assessed in rat liver by double immunohistochemical staining using PC10 and anti-BrdU monoclonal antibodies to identify labelled nuclei of parenchymal and sinusoidal cells. In formaldehyde-fixed human biopsies (n = 13) PCNA-labelling index (PCNA LI) was 0.43 +/- 0.24% (mean +/- SEM) for hepatocytes and 0.09 +/- 0.03% for sinusoidal cells. A great interspecimen variability was observed and a preferential lobular distribution was not evident. In methanol-fixed human liver (n = 8) the immunostaining was strong. PCNA LI was 0.05 +/- 0.01% for hepatocytes and 0.14 +/- 0.01% for sinusoidal cells. 75% of labelled hepatocytes and 60% of labelled sinusoidal cells were found in acinar zone 1. In formaldehyde-fixed rat liver (n = 10) a weak nuclear staining and a great interspecimen variability were evident. LI was 0.13 +/- 0.07% for hepatocytes and 0.40 +/- 0.21% for sinusoidal cells without preferential acinar distribution. In methanol-fixed rat liver (n = 10), PCNA LI was 0.14 +/- 0.02% for hepatocytes and 0.40 +/- 0.04% for sinusoidal cells. 64% of labelled hepatocytes and 50% of labelled sinusoidal cells were found in zone 1. Only on methanol-fixed material did double immunohistochemistry show an almost complete overlap of BrdU and PCNA labelling. The PCNA LIs and the zonal distribution of labelled nuclei as obtained in methanol-fixed material are in keeping with previous reports using 3H-thymidine (3H-Thy) incorporation, suggesting that PCNA immunostaining represents a valid alternative to 3H-Thy.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic ethanol (EtOH) metabolism has been assumed to involve hepatocytes differently, according to their location in the hepatic acinus. This study's aim was to gain information on plasma membrane (PM) order parameter in periportal (PP) and perivenular (PV) hepatocyte-enriched fractions isolated by a digitonin-collagenase perfusion technique from rats pair-fed for 6-8 wk liquid diets containing either EtOH or isocaloric carbohydrates. Fluorescence polarization (P) studies have been performed to measure PM order parameter of PP and PV hepatocytes cultured for 2-6 h on glass cover slips and labeled with 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific probe for PM of living cells. Fluorescence polarization and microscopy indicated that TMA-DPH is a suitable probe to study PM order parameter in subconfluent rat hepatocyte monolayers where it labeled, after a rapid incorporation, PM of cells. In pair-fed control rats, PM order parameter was lower in PP hepatocytes than in PV cells (P = 0.366 +/- 0.013 vs. 0.381 +/- 0.021, respectively, P < 0.02; n = 7). In EtOH-treated rats, these zonal differences tended to disappear (P = 0.419 +/- 0.012 in PP cells vs. 0.417 +/- 0.007 in PV cells; n = 7). In addition, the order parameter was significantly higher either in PP or PV hepatocytes compared with pair-fed control animals (P < 0.002 and 0.003, respectively). A 30-min culture of cells in the presence of 40-200 mM EtOH significantly decreased the PM order parameter of hepatocytes isolated from pair-fed control rats with respect to EtOH-treated animals both in PP and PV cells (P < 0.01 and 0.02, respectively; n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Liver acinus shows a well-known metabolic zonation. The aim of this study was to investigate intracellular pH (pHi) regulation in isolated periportal (PP) and perivenular (PV) rat hepatocytes., Methods: 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein was used as pH-sensitive dye. Hepatocyte subconfluent monolayers were acid-loaded by exposure to 20 mmol/L NH4Cl and alkali-loaded by reducing external CO2 and HCO3- at an external pH of 7.4., Results: In the presence of HCO3-/CO2, (1) baseline pHi was higher in PP (7.25 +/- 0.018) than in PV hepatocytes (7.20 +/- 0.013) (P < 0.05); (2) pHi recovery from an acid load was 40% higher in PP than in PV hepatocytes (P < 0.02) and was inhibited by amiloride by 36% in PV and 7% in PP hepatocytes; (3) DIDS inhibited amiloride-independent pHi recovery from an acid load by 65% in PP and 52% in PV cells. In the absence of HCO3-/CO2, baseline pHi and pHi recovery from an acid load were not significantly different in PP and PV hepatocytes. pHi recovery from an alkali load was 30% higher in PV than in PP cells (P < 0.02)., Conclusions: Our data suggest that isolated PP rat hepatocytes show higher activity for Na(+)-HCO3- cotransport, whereas PV cells show greater activity for Cl-/HCO3- exchanger.

Proliferating lipocytes (fat-storing cells or perisinusoidal stellate cells of the liver) were detected by in vivo incorporation of bromodeoxyuridine (BrdU) in an experimental model of cirrhosis in the rat by dimethylnitrosamine. Lipocytes were identified by sequential double immunohistochemical staining on frozen sections using anti-desmin antibodies as a marker of cytoplasmic intermediate filaments followed by anti-BrdU antibodies to identify S-phase nuclei in animals treated for 7, 14 or 21 days. The number of desmin-positive (lipocytes) and desmin-negative (Kupffer and endothelial cells) sinusoidal cells incorporating BrdU was recorded. The labelling index of lipocytes was calculated as the percentage of BrdU-labelled desmin-positive cells with respect to total number of lipocytes. In control animals, when the total number of lipocytes was 153.9 +/- 11/mm2 (mean +/- 1 S.E.) the number of desmin-positive S-phase sinusoidal cells never exceeded 6.8 +/- 1.2/mm2 with a maximum labelling index of 4.3 +/- 0.5%. At 7 days of treatment, the values were respectively 236 +/- 26.5/mm2, 53.2 +/- 5.9/mm2 and 22.6 +/- 0.5% (p less than 0.001 vs. controls), while, at 21 days they were 272.5 +/- 21.2/mm2, 23.3 +/- 4.0/mm2 and 8.5 +/- 1.1% respectively (p less than 0.01). These results show that hyperplasia of lipocytes represents an early reaction to dimethylnitrosamine-induced liver injury. The local accumulation of lipocytes appears to occur in areas where fibrous septa develop later on.

The present study deals with a case of hepatic parenchymal steatosis in a child with primary ciliary dyskinesia (immotile cilia syndrome) well documented by functional and ultrastructural evaluation of the ciliary epithelia. Hepatic steatosis was associated with ultrastructural evidence of retention of material either in the cisternae of the endoplasmic reticulum or in proximity of the Golgi apparatus of hepatocytes. It is suggested that the absence of dynein in the axoneme is probably part of a diffuse genetic defect which may extend to cytoplasmic, non axonemal, dynein and lead to a disturbance of various microtubule-dependent cell activities.

The present study is concerned with the immunohistochemical characterization in situ of the mononuclear infiltrate accompanying the formation of septa and the development of cirrhosis in the liver of rats treated with dimethylnitrosamine (DMN), i.p. (10 microliters/kg, 3 days a week for 3 weeks). Monoclonal antibodies against macrophages, pan T cells, T cell subsets and B cells have been applied on cryostat sections of animals given DMN for 7, 14 and 21 days. The maximum increase of macrophages and lymphocytes was observed at days 7 and 14 respectively. At all times T lymphocytes appeared as the major component of the inflammatory infiltrate with a largely predominant population of cytotoxic/suppressor T cells. At day 21, with evidence of nodulation of parenchyma, macrophages levelled off while T cells remained numerous without changes in the inducer-helper T cells/cytotoxic-suppressor T cells ratio which remained always less than 1. B cells were always few. These findings illustrate the early influx of lymphocytes in DMN-induced liver injury and help to define the lymphocyte subsets associated with inflammation and fibrosis in a reproducible animal model.

The visualization of incorporation sites of the thymidine analog bromodeoxyuridine (BrdU) into DNA, detected by immunocytochemistry, has been proposed as an index of the percentage of S-phase cells in a variety of tissues and as an easy, less expensive alternative to autoradiography. This technique has not yet been applied to the study of physiological cell renewal in the normal liver. In the present study, results obtained with this method in the liver of normal young adult rats is reported. BrdU was administered in vivo and subsequent incorporation was detected by the PAP technique using monoclonal anti-BrdU antibodies. The nuclei exhibiting a positive reaction within the liver were few and accounted for about 0.45% of all hepatocytes. Positive cells were located preferentially in zone 1, which contained 82.7% of the labeled cells. Zone 2 contained 15.4%, while only 1.9% of the labeled cells were found in zone 3. Positive-staining Kupffer cell nuclei were rare (about 0.5% of all Kupffer cells) and were distributed randomly in the hepatic lobule. These findings provide quantitative data about hepatocyte renewal in the normal liver in the absence of a growth stimulus. The simplicity and the reproducibility of this technique suggests that further application of this method in situations assessing hepatic regeneration are indicated.

The present study is concerned with changes in the number and localization of S-phase cells in the liver of rats exposed to dimethylnitrosamine (DMN). S-phase cells were detected by immunohistochemistry after injection of bromodeoxyuridine (BrdU) and exposure of paraffin sections of liver tissue to the antibody anti-BrdU. With respect to controls, the number of S-phase cells increased four to fivefold in DMN-treated animals in the first week of treatment and remained significantly higher thereafter, in association with the formation of septa. At all times, the labelling index was higher in littoral cells than in hepatocytes. No labelling was observed in biliary cells. This behaviour is different from that reported in other situations, for instance in regeneration after partial hepatectomy, which suggests that besides hepatocytes and littoral cells replacement, an involvement of the latter cell line in the inflammatory reaction, synthesis of extracellular matrix components and formation of septa may account for this particular pattern.

Liver fibrosis was induced in rats after administration of dimethylnitrosamine (DMN) intraperitoneally three times a week for 3 weeks. Incomplete septa appeared after 7 days and evidence of nodulation of the parenchyma was observed after 21 days. Both distribution of extracellular matrix components (collagen type I, type III and type IV, laminin, fibronectin, heparan sulphate proteoglycan) and the distribution of desmin as a marker of lipocytes (Ito cells) and of iso-alpha-smooth muscle actin were studied with immunoperoxidase. Changes in the distribution of extracellular matrix components outlined both the formation of septa and the development of nodules with changes in the sinusoidal pattern evoking aspects of capillarization. The number of desmin-positive cells increased in DMN-treated animals, showing a prominent reaction in the fibrous septa. In the normal liver, lipocytes were positive for laminin and negative for actin, but septal and juxta-septal lipocytes were positive for both antigens, suggesting the presence of transitional cells with mixed immunoreactivity. This was confirmed by ultrastructural studies which showed typical intraseptal myofibroblasts and other elements exhibiting the structural features of both myofibroblasts and lipocytes.

The role of microtubules on membrane fluidity has been investigated on freshly isolated whole rat hepatocytes prepared by the perfusion method and exposed either to the microtubule-depolymerizing drugs colchicine and vincristine or to beta-lumicolchicine, a colchicine analog deprived of biological activity. Exposure of hepatocytes to 6.3 microM colchicine or to 3.0 microM vincristine led to a significant decrease of membrane fluidity as measured by fluorescence polarization of trimethylammoniodiphenylhexatriene (TMA-DPH). No changes were observed in cells exposed to 10.0 microM beta-lumicolchicine. These observations support the hypothesis that the microtubular system plays a role in the modulations of physico-chemical properties of the plasma membrane.

The study of membrane fluidity is a rapidly expanding field of research and its interest in hepatology has been stressed recently. The present study is the first report concerned with the determination of membrane fluidity of isolated rat hepatocytes. The data have been compared with those obtained in plasma membrane fractions and subfractions (basolateral or canalicular) derived from homogenates. The fluorescent probes used to measure the fluidity were diphenylhexatriene (DPH) a 'classical' probe, and its derivative trimethylammoniodiphenylhexatriene (TMA-DPH) at 25 degrees C and at 37 degrees C. The values obtained with DPH were lower than those with TMA-DPH, probably due to the localization of the probes in different regions of the phospholipid bilayer. In addition, DPH revealed significant differences in the fluorescence polarization values obtained in isolated hepatocytes compared with membrane fractions, which was in contrast to TMA-DPH, where the respective values were of the same order of magnitude. This behaviour is probably due to the mobility of DPH in the membrane core and its rapid internalization into the cell, whereas TMA-DPH remains anchored for a long time on the cell surface. These findings suggest that TMA-DPH is a better probe than DPH for measuring the fluorescence polarization of whole isolated hepatocytes and that the use of different probes might be of help in exploring different zones of the membrane bilayer.

Valproic acid (VPA) is a simple fatty acid largely used as anticonvulsivant agent. Side effects are uncommon, but cases of fatal hepatic failure have been reported. To elucidate the mechanism of VPA-induced hepatotoxicity, the functional and structural changes associated with administration of sodium valproate (NaVPA) to rats (200 or 600 mg per kg, i.p.) were analyzed. NaVPA produced an immediate, dose-dependent and prolonged increase in bile salt-independent bile flow with a decrease in biliary cholesterol and phospholipid output. At 3 and 5 hr, marked ultrastructural changes were evident in hepatocytes, including formation of autophagic vacuoles engulfing altered mitochondria and occasionally peroxisomes. A modest accumulation of lipoprotein particles was evident at 5 hr in the Golgi cisternae. Twelve-hour samples appeared normal. Bile canaliculi and junctional complexes remained unaltered throughout. The changes observed differ from those previously reported with other hydrocholeretics, such as diethylmaleate; they are likely related to hepatic biotransformation of VPA, which undergoes beta and omega-oxidation, and glucuronidation. While VPA-induced choleresis reflects the physiological osmotic effect of the glucuronide excreted in bile, the ultrastructural changes likely reflect interference by VPA with beta-oxidation of endogenous fatty acids and temporary accumulation of transformation products in the mitochondrial matrix.

The livers of rats given ethanol for 5 weeks showed marked structural alterations of hepatocytes of acinar zone 3 including mitochondrial pleomorphism, increased smooth endoplasmic reticulum and deposition of small (less than 0.5 micron) lipid droplets. In addition, apoptotic bodies involving altered parenchymal cells were frequently observed, together with prominent mononuclear infiltrates adjacent to the terminal hepatic veins. It is suggested that 'age' of liver cells may play a role in the preferential perivenular localization of early ethanol-induced liver damage.

The aim of the present investigation was to study the number and acinar distribution of apoptotic bodies in normal liver as an approach to a better understanding of cell kinetics in the hepatic parenchyma. The material included 20 normal human needle liver biopsies and 20 normal male Sprague-Dawley rats. For each liver sample, the following parameters were measured: number of apoptotic bodies in the lobule, topographical localization, and distance from terminal hepatic veins (THV), i.e., the row of hepatocytes concerned, H1 being the closest to the THV. The results were strikingly similar in human and in animal material, showing that apoptotic bodies are rare in the normal liver and, when present, are always observed in zone 3, next to the THV. In fact, the first two rows of hepatocytes (H1 and H2) contained 80% of the apoptotic bodies in human liver, and 95% in rat liver. These data show that apoptotic bodies are not randomly dispersed in normal liver tissue but show a preferential acinar distribution. In addition, the vast majority of apoptotic bodies are located in the row of liver cells immediately adjacent to the THV. If apoptosis is indeed an expression of physiological cell renewal or 'programmed cell death', these findings support the concept of an aging gradient of liver cells, with zone 3 containing older hepatocytes.

The aim of the present study was to determine if data on the number and acinar distribution of apoptotic bodies (AB) in normal liver could help in the understanding of cell kinetics in the liver, and the mechanism of early ethanol-induced liver damage. Normal male Sprague-Dawley rats were studied. They had free access to Purina chow and drinking water. Ethanol-treated rats received the drug at increasing concentration in drinking water for 5 weeks. The following parameters were measured: number of AB in the lobule, topographical localization, distance from terminal hepatic veins (THV), i.e. row of hepatocytes concerned, H1 being the closest to the THV. The results show that AB are rare in the normal liver and are always observed in zone 3, next to the THV. Of 149 THV examined, 56 showed one associated AB, exceptionally two. 74% of the AB were confined to the first row of hepatocytes (H1), 21% to H2, 4% to H3, and 1% to H4. In ethanol-treated rats the mean number of AB was 2 or 3 for each THV. 42% were found in H1, 32% in H2, 15% in H3, 7% in H4, and 4% in H5. The data show that AB are not randomly dispersed in normal liver but show a preferential acinar distribution. In addition, most AB are located in the row of liver cells immediately adjacent to the THV. If apoptosis is indeed an expression of physiological cell renewal, these findings support the concept that zone 3 contains older hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Horseradish peroxidase (HRP) and lanthanum chloride (LaCl3) are useful tools for, respectively, the study of vesicular transport through the hepatocyte and the study of the permeability of junctional complexes. These tracers have been used to detect the changes associated with the choleresis independent of bile acids induced by valproic acid (VPA) in rats. The animals were given a single dose of VPA (600 mg/kg, ip). HRP (100 mg/kg) or 5 mM LaCl3 were given intraportally after 1 h, when bile flow had increased twofold. The excretion of HRP in bile was measured colorimetrically up to 2 h after HRP. Ultrastructural morphometry was conducted on liver of intact rats taken from 1 to 40 min after HRP. The volume density (VD) of HRP-containing vesicles and of HRP-containing multivesicular bodies (MVB) was counted. In VPA-treated rats, HRP appeared in bile with a peak showing at 5 min against 20 min in controls, but the total amount of HRP excreted was less than in controls. The intrahepatocytic vesicular transport of HRP was also modified, showing a peak at 3 min in VPA-treated rats compared to 10 min in controls, together with a decreased VD of pericanalicular vesicles. This was accompanied by an increase of HRP-containing MVB, already evident at 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative analysis of needle liver biopsies of patients with untreated, uncomplicated cholelithiasis has been performed to better evaluate eventual changes occurring under medical therapy of gallstones. With the light microscope, the intensity of parenchymal steatosis was variable among the patients but the mean volume density of lipid droplets was significantly increased as compared to normal subjects. In addition, there was a significant increase in the volume density of lipocytes (fat-storing cells) without an increase in the number per square area, suggesting a hypertrophy of individual cells. The data obtained by ultrastructural morphometry show a significant increase in the surface density of the rough endoplasmic reticulum in the hepatocytes, a significant increase of the mitochondrial volume density together with the presence of curled mitochondrial cristae and a slight, inconstant intracellular and intracanalicular bile retention. No qualitative or quantitative changes were observed at the level of the smooth endoplasmic reticulum. The activity of the microsomal enzyme NADPH-cytochrome c reductase as evaluated on liver biopsy material was in the normal range. Some of the features observed in this group of patients are reminiscent of alterations previously described in human or experimental cholestasis and suggest that they might depend on a common underlying disturbance in cholesterol and bile salt metabolism.

The hepatic synthesis of cholesterol-measured as incorporation of 14C-acetate into digitonin-precipitable sterols-has been evaluated in needle biopsy material of normal untreated patients and patients under short-term treatment with Diazepam, Phenobarbital, Chlorpromazine or Diphenylhydantoin. A significant increase of cholesterol synthesis was observed in the first two groups with much higher levels of incorporation in the Diazepam-treated patients. Moreover in this group the levels were still elevated from 4 to 7 days after drug withdrawl. Ultrastructural and morphometric studies performed on the same biopsy material showed a significant increase of smooth endoplasmic reticulum in hepatocytes of Diazepam-treated patients; in addition, there seemed to be a positive correlation between the increased cholesterol synthesis and the formation of areas of non vesicular, type 2, smooth endoplasmic reticulum. These findings suggest an early stimulation of the liver cell microsomal system by Diazepam in man; they also point to side effects of some drugs, which are not predictable from studies in Wistar rats.

A quantitative analysis of lipocytes (fat-storing cells, Ito cells) has been conducted on needle liver biopsies of 8 subjects without hepatic impairment. The mean volume density of lipocytes was 1.88 +/- 0.25% of parenchymal volume, and the number of lipocytes per 1,000 hepatocytes (Ito cell index) was 63.45 +/- 19.18. A study of the zonal distribution showed that the values were significantly higher in the centrolobular than in the periportal areas. No correlation was evident between the volume density or the number of lipocytes and the volume density of parenchymal steatosis. The high coefficients of variation resulting from the present study are evidence that quantitative data are necessary to evaluate volumetrical or numerical variations of lipocytes.

The distribution of the endoplasmic reticulum in human hepatocytes is defined in quantitative terms using the techniques of morphometry. The subjects of the study are liver biopsies from normal, untreated subjects and patients being treated with various drugs. In contrast to rat hepatocytes, the amount of smooth endoplasmic reticulum (SER) in man exceeds that of the rough endoplasmic reticulum (RER) and accounts for 76.3% of the total endoplasmic reticulum. This is to be taken into consideration in pharmacological or toxicological studies. In addition, two components of the SER have been identified: more prominent is the type 1 or vesicular which has a regular honeycomb pattern, made up of cisternae with patent lumina and a mean width of 1500 A; the type 2, or non-vesicular, occurs in discrete foci of densely packed smooth membranes with a spacing of about 140 A. In subjects under short-term treatment with Benzodiazepin (diazepam) the RER remained unchanged but the SER membranes were significantly increased with a remarkable, two-to threefold increase of the SER type 2 in three out of four patients. A rise in incorporation of (14)C-acetate into digitonin-precipitable sterols as measured in liver biopsy material was also noted in these three patients. The suggestion is made that the SER 2 represents the newly formed membranes whereas the SER 1 would represent ;adult' membranes. No changes were observed in two patients under short-term treatment with phenobarbital or Dilantin.

Hepatic fibrosis has been induced in rats by low doses of dimethylnitrosamine (DMN) and special attention has been paid to early morphological events. DMN (10 microliter/kg body wt., i.p.) was given 3 days a week for 3 weeks to Sprague-Dawley rats. Liver samples were taken on days 7, 14, 21, 28 and 35 and examined by light and electron microscopy. Hemorrhagic necrosis, mainly centrolobular, was evident on day 7, with disruption of the sinusoidal lining, and widening or disappearance of the spaces of Disse invaded by erythrocytes and lymphocytes. Large granular lymphocytes similar to pit cells were also present in close contact with hepatocytes. At day 14, fibrotic septa were associated with cells bearing 'transitional' features between those of lipocytes, myofibroblasts and fibroblasts. Hepatocytes showed foci of increased smooth endoplasmic reticulum, and altered sinusoidal and canalicular membranes. At day 21, all animals showed nodularity of the parenchyma, with evidence of micronodular cirrhosis associated with ascites in two animals. At day 35 (19 days after cessation of treatment) there was little residual inflammation, but well-defined micronodules were still present in all animals. This shows that, in the rat, 3-week treatment with a low dose of DMN produces micronodular cirrhosis following diffuse hemorrhagic necrosis without steatosis. The response of the animals was uniform and reproducible. Lesions of the sinusoidal wall and of membranes of liver cells associated with the inflammatory reaction appeared prominent.

The influence of sex and age on membrane fluidity, has been investigated in 6, 12, 18 weeks old Sprague-Dawley rats. Fluorescence polarization (P) was determined at 37 degrees C with a Perkin Elmer MPF 44A fluorescence spectrophotometer. The fluorescent probe TMA-DPH was added to isolated hepatocytes prepared by collagenase method. The membrane fluidity was constantly lower in males than in females, but the difference was statistically significant only in the 12 weeks old group. Major differences appeared related to aging with a significant age-related decrease in fluidity in all animals.

Chenodeoxycholic acid (CDCA) is an effective treatment for dissolving gallstones but experimental studies have suggested that it might be hepatotoxic. The present study is concerned with a group of patients undergoing medical therapy for gallstones for periods of 30 days up to 14 months with CDCA (15 mg/kg/day). Routine functional tests, determination of some liver microsomal enzymes and stereological studies of the liver tissue have been performed and the data have been compared with those obtained before treatment. No significant changes were observed in the functional tests throughout the study. Also the microsomal mixed function oxidase system seemed unaffected by CDCA therapy. The histological features of the liver biopsies were not appreciably different from those observed prior to treatment. Although there were large interindividual variations, the volume density of parenchymal steatosis and of the lipocytes remained comparable in the ssme individual. The ultrastructural features noted in untreated subjects such as curled mitochondrial cristae, slight intracellular bile retention, increased surface density of the rough endoplasmic reticulum were still evident after 14 months of treatment. No additional changes were noted. These results show that no evidence of hepatotoxicity seems to develop in man under therapy with CDCA at the dose considered. But the structural abnormalities observed before treatment appear to persist even in subjects under long-term therapy.

Ultrastructural studies of the liver of rats given diethylmaleate (DEM) (0.7 ml per kg body, i.p.) weight revealed marked alterations of the configuration of the Golgi complex in all hepatocytes. This consisted in dilatation of the cisternae leading to the formation of large cysts. The alteration was more prominent 15 min after administration of the drug, coincident with a 2-fold increase of bile flow and a decrease of hepatic glutathione to 15% of controls. The morphology returned to normal at 45 min after DEM administration together with a return of bile flow to control values whereas hepatic glutathione level remained markedly depressed for 2 hr, rising slowly afterwards. No other signs of altered hepatic morphology developed up to 5 hr. The morphological changes correlated with altered bile flow induced by DEM, suggesting involvement of the Golgi complex in biliary excretion of osmotically active glutathione conjugates of DEM.

The study is concerned with the effects of cimetidine (C), ranitidine (R) and oxmetidine (O) on rat liver cells, in order to compare the effects of increasing amounts of the drugs on various metabolic pathways and on hepatocyte morphology, and their relationship with the drug structure, namely the presence of an imidazole or furan ring. In vitro, an inhibition of ethylmorphine N-demethylase (E-DM) (60%) and of aniline hydroxylase (A-OH) (20%) was observed when either C or R were added at a concentration higher than 0.5 mM to microsomes from untreated rats. Microsomes from phenobarbital or 3-methylcholanthrene-pretreated animals showed a 30 to 40% inhibition of E-DM (mixed type inhibition). In vivo, administration of a single dose (475 microM/kg) of R was followed by an inhibition of E-DM activity, significantly less pronounced (p less than 0.05) than following an equimolar amount of C, whereas O did not alter the enzymatic activity. The amount of cytochrome P-450 and the activities of NADPH cytochrome c reductase and of A-OH were substantially unaffected. No changes of smooth endoplasmic reticulum or of other liver cell organelles were observed by electron microscopy. These findings demonstrate that both in vivo and in vitro E-DM appears as the more sensitive among the pathways considered. Moreover the data support the view that the mere presence of the imidazole ring does not account for the inhibitory effect of H2 antagonists.

Rifampicin induces a proliferation of the smooth endoplasmic reticulum in guinea-pig and human hepatocytes. This may support the hypothesis of enhancement of drug-metabolizing enzymes induced by the drug. However, the pattern of proliferation is not similar in man and in guinea-pig hepatocytes. Some caution is needed in the study of enzyme induction in man and in extrapolations from animal to human data.