The antiestrogen toremifene protects against alcoholic liver injury in female rats.

BACKGROUND/AIMS: Females are generally considered to be more susceptible to alcohol-induced liver injury than males. To elucidate whether gonadal hormones are involved, female rats were chronically treated with ethanol and with an antiestrogen. METHODS: Ethanol was administered in a low-carbohydrate liquid diet. Estrogen action was blocked by daily intubation of toremifene, a non-hepatotoxic second generation estrogen receptor antagonist. RESULTS: The female rats consuming intoxicating amounts of ethanol diet for 6 weeks developed massive microvesicular/macrovesicular steatosis, frequent inflammatory foci and spotty necrosis. Serum alanine aminotransferase increased 7-fold. Toremifene treatment did not affect steatosis, but significantly reduced inflammation and necrosis. Ethanol increased the expression of CD14 and tumor necrosis factor- (TNF) alpha mRNA and also the production of TNF-alpha by isolated Kupffer cells, but toremifene had no significant counteracting effect. However, toremifene significantly alleviated both ethanol induction of the pro-oxidant enzyme CYP2E1 and ethanol reduction of the oxidant-protective enzyme Se-glutathione peroxidase. CONCLUSIONS: The partial protection by toremifene against ethanol-induced liver lesions suggests a pathogenic contribution of estrogens, possibly associated with an oxygen radical mediated mechanism.

Kupffer cell inactivation alleviates ethanol-induced steatosis and CYP2E1 induction but not inflammatory responses in rat liver.

BACKGROUND/AIMS: Gadolinium chloride inactivates Kupffer cells and alleviates alcohol-induced liver lesions. We investigated the mechanism of gadolinium chloride protection after oral ethanol feeding. METHODS: Rats were maintained ethanol-intoxicated for 6 weeks by feeding ethanol in a low-carbohydrate/high-fat liquid diet. Macrophages were inactivated by intravenous administrations of gadolinium chloride. At termination, liver samples and cell lysates obtained from the periportal and perivenous region were analyzed for histopathology, mRNA expression of endotoxin-associated parameters and cytokines and for enzymes involved in oxidative stress. RESULTS: Ethanol treatment alone caused marked microvesicular/macrovacuolar steatosis and focal inflammation. Gadolinium significantly alleviated pathology, by reducing steatosis but not inflammation. Gadolinium treatment eliminated ED2 immunopositive Kupffer cells, which were larger and more frequent periportally. Ethanol significantly increased the mRNA expression of the endotoxin (LPS) receptor CD14 and the LPS binding protein LBP, but not that of the pro-inflammatory cytokines TNF-alpha and IL-1beta. The mRNA of CD14 was found to be expressed preferentially in the perivenous region, but gadolinium treatment had no significant effect on the expression or the distribution. However, gadolinium significantly moderated the ethanol induction of CYP2E1 and this effect correlated to the degree of steatosis. Ethanol increased glutathione transferase and reduced glutathione peroxidase activity, but these changes persisted after gadolinium treatment. CONCLUSIONS: Our results suggest that gadolinium chloride reduces symptoms of ALD mainly by counteracting steatosis, and that CD14-positive Kupffer cell populations are not involved in gadolinium protection. The strong correlation between pathology and CYP2E1 induction might suggest a steatopathogenic role for this enzyme.

A web-based data collection system for clinical studies using Java.

Data collection in multi-centre controlled clinical trials is a major problem. We describe a web-based documentation system for data collection via the Internet we have developed in Java. It keeps both the platform independent program (a Java applet) and the database on a single server to which the participating centres have access at any time. It is based on a three-tier model where client requests are handled and monitored by an application server (a Java application). We show the advantages such a system has over the HTML/CGI combination which is often used for database access, as well as possible extensions, e.g. connection to a data dictionary. Finally, we discuss the security problems which arise by the use of the Internet for data collection.

Aryl hydrocarbon receptor-associated genes in rat liver: regional coinduction of aldehyde dehydrogenase 3 and glutathione transferase Ya.

The tumor-associated aldehyde dehydrogenase 3 (ALDH3) and the glutathione transferase (GST)Ya form are coded by members of the Ah (aryl hydrocarbon) battery group of genes activated in the liver by polycyclic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The physiological role of the Ah receptor (AHR), its gene-activating mechanism and its endogenous ligands are still poorly clarified. We had previously observed that 3-methylcholanthrene (3MC) and beta-naphthoflavone (betaNF) induced the AHR-associated CYP1A1/1A2 pair in different liver regions, an effect not explained by the acinar distribution of the AHR protein. Here, we investigated AHR-associated regional induction by comparing the expression patterns of ALDH3 and GSTYa. Analysis of samples from periportal and perivenous cell lysates from 3MC-treated animals revealed that ALDH3 mRNA, protein and benzaldehyde-NADP associated activity were all confined to the perivenous region. In contrast, such regio-specific induction was not seen after beta-NF induction. Immunohistochemically, a peculiar mono- or oligocellular induction pattern of ALDH3 was seen, consistently surrounding terminal hepatic veins after 3MC but mainly in the midzonal region after betaNF. A ligand-specific difference in regional induction of GSTYa1 mRNA was also observed: The constitutive perivenous dominance was preserved after 3MC while induction by betaNF was mainly periportal. A 3MC-betaNF difference was also seen by immunohistochemistry and at the GSTYa protein level, in contrast to that of the AHR-unassociated GSTYb protein. However, experiments with hepatocytes isolated from the periportal or perivenous region to replicate these inducer-specific induction responses in vitro were unsuccessful. These data demonstrate that the different acinar induction patterns by 3MC and betaNF previously observed for CYP1A1 and CYP1A2 are seen also for two other Ah battery genes, GSTYa1 and ALDH3, but in a modified, gene-specific form. We hypothesize that unknown protein(s) operating in vivo and modifying the Ah-mediated response at the common XRE element located upstream of these genes is affected zonespecifically by 3MC and betaNF.

Data collection in multi-center clinical trials via Internet. A generic system in Java.

Data collection via Internet is usually performed with an HTML/CGI combination, which has a lot of disadvantages, most important the lack of security features. We therefore have developed a system written entirely in Java, which implements a true client/server application based on TCP/IP. The documents are created using a multi-lingual data dictionary, and the used GUI components are able to perform plausibility checks, which improves quality of the data. The system is designed to be easily extensible so that it can be used in almost any kind of clinical trials. It is based on a three-tier model where client requests are handled and monitored by an application server. We will describe this system and it's implementation and compare it to the HTML/CGI approach. Of special interest are security features, which are possible through the use of Java.

A data dictionary approach to multilingual documentation and decision support for the diagnosis of acute abdominal pain. (COPERNICUS 555, an European concerted action).

This paper describes the design and development of a multilingual documentation and decision support system for the diagnosis of acute abdominal pain. The work was performed within a multi-national COPERNICUS European concerted action dealing with information technology for quality assurance in acute abdominal pain in Europe (EURO-AAP, 555). The software engineering was based on object-oriented analysis design and programming. The program cover three modules: a data dictionary, a documentation program and a knowledge based system. National versions of the software were provided and introduced into 16 centers from Central and Eastern Europe. A prospective data collection was performed in which 4020 patients were recruited. The software design has been proven to be very efficient and useful for the development of multilingual software.

Collection of data in clinical studies via Internet.

This paper describes a system enabling data collection in multicenter clinical trials via WWW and Internet. The form-based data entry is based on HTML documents with JavaScript linked to a relational database (mSQL) via a cgi program (w3-msql). The design has been applied to a multi-national study in acute abdominal pain, for which eight clinical forms have been developed. The system is now in test use and experiences with this approach are presented.

Distribution of glutathione S-transferase isoforms in rat liver after induction by beta-naphthoflavone or 3-methylcholanthrene.

Regional differences in vulnerability to xenobiotic liver damage may relate to the distribution of the detoxication capacity of the glutathione S-transferases (GST). HPLC analysis of cell lysates obtained by digitonin infusion from either the periportal or the perivenous region revealed that the content of all the GST subunits investigated (1, 2, 3, 4 and 8) was higher in the perivenous region. The strongest perivenous dominance was observed for subunit 1 (Ya) and the alpha class appeared to be more zonated that the mu class. A similar perivenous dominance was observed by analysis of GST activity with either 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloronitrobenzene (DCNB) or trans-4-phenyl-3-buten-2-one (PBO) as substrate. In contrast, with cumene hydroperoxide (CuOOH) or tert-butyl hydroperoxide (tBOOH) as substrate a reciprocal twofold periportal dominance was observed. Induction by pretreatment with beta-naphthoflavone reduced or abolished the perivenous dominance of the alpha-subunits 1, 2 and 8. In contrast, after pretreatment with 3-methylcholanthrene, only the acinar gradient of subunits 2 (Yc) was abolished, while the strong perivenous gradient subunit 1 (Ya) was maintained and that of subunit 8 (Yk) increased. CDNB based assays demonstrated that beta-naphtoflavone treatment reduced (from 2.1 to 1.4) while 3-methyl cholanthrene enhanced (to 2.6) the perivenous/periportal GST activity ratio. Assays based on CuOOH or tBOOH indicated that neither the Se-dependent nor the Se-independent glutathione peroxidase activity nor its acinar distribution was affected by the inducers. These results demonstrated that although the expression of all investigated members of the alpha and mu classes is higher in the perivenous region, there are marked isozyme differences, the acinar gradient being particularly prominent for subunit 1 (Ya). The distinct difference in the acinar induction pattern of GST Ya between beta-naphthoflavone and 3-methylcholanthrene resembles that reported for cytochrome P450 (CYP1A1 and CYP1A2), also members of the aryl hydrocarbon (Ah) receptor genes, suggesting common regionally acting regulatory elements in the expression of these genes in the liver.

Protective effect of various calcium antagonists against an experimentally induced calcium overload in isolated hepatocytes.

The effect of the hepatotoxic substance diamidinothionaphthene (98/202) on cytosolic, mitochondrial and extra-mitochondrial calcium distribution was measured in isolated rat hepatocytes. The drastic disturbance of the intracellular calcium homeostasis caused by this substance (increase of the cytosolic and mitochondrial calcium contents and depletion of extra-mitochondrial calcium stores, which at last lead to cell death) gave rise to an investigation of the possible cytoprotective effect of calcium antagonists of various chemical classes: verapamil, diltiazem, and nifedipine on isolated hepatocytes. Our results show that all three calcium antagonists prevented cell death caused by 98/202. The 98/202-induced increase of cytosolic and mitochondrial calcium content was inhibited by all three calcium antagonists. However, only verapamil was able to inhibit the depletion of extra-mitochondrial calcium stores. Since 98/202-induced cell death occurs only in the presence of extracellular calcium, it is concluded that calcium antagonists are also able to inhibit the influx of extracellular calcium in liver cells, which leads to a calcium overload of the cytosol and mitochondria. The various ways of interfering with the calcium homeostasis of liver cells qualifies the hepatotoxic substance 98/202 as a suitable in vitro hepatotoxicity model for testing the hepatoprotective effect of different calcium antagonists.

Influence of pentamidine and two new trypanocidal agents (DAPI, DIPI) on liver metabolism of mice.

The effects of equimolar doses (65.5 mumol/kg intraperitoneally) of three trypanocidal compounds, i.e., pentamidine, 6-amidino-2-(amidinophenyl)indole (DAPI), and 6-imidazolino-2-(imidazolinophenyl)indole (DIPI) on some parameters of liver carbohydrate, lipid and energy metabolism have been assessed in male NMRI mice. Most prominent effects were an initial increase of the blood glucose and fatty acid levels followed by long lasting increases of the hepatic triglyceride and glycogen contents which were accompanied by decreases of the liver pyruvate and lactate and ATP contents. These effects which can be interpreted as results of a transient lipolytic and glycogenolytic effect and a longer lasting inhibition of the energy yielding carbohydrate metabolism were most pronounced after DAPI and DIPI and less marked after pentamidine.

Regioselective induction of liver glutathione transferase by ethanol and acetone.

Both ethanol and acetone are substrates and inducers of the cytochrome P450 IIEI. This isoenzyme is induced in the perivenous region, which may explain the centrilobular damage elicited by several hepatotoxins being substrates for P450 IIE1. Here we demonstrate that induction of glutathione S-transferase after ethanol and acetone treatment is also restricted to the perivenous region, suggesting regiospecific enhancement of the transferase associated cellular defence capacity. The total glutathione peroxidase activity does not increase after the induction.

Investigations on mutagenicity and genotoxicity of pentamidine and some related trypanocidal diamidines.

Pentamidine, DAPI and some related compounds (DAI, 6-Br-AI, DPTN, DIPI, 3-Am-DAI, DiaPBF) were investigated in 2 different screening test systems for their potential mutagenic and cytotoxic effects, in the light of their binding to DNA. In the Ames test using Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation no mutagenic effects could be observed. All diamidines tested, except DAI, were toxic at concentrations of 0.5 and 1.0 mumole/plate. In the sister-chromatid exchange (SCE) assay with human peripheral lymphocytes all compounds tested were growth-retarding particularly in the G0 phase. A significant induction of SCEs could only be seen after treatment with the monoamidino compound 6-Br-AI at a concentration of 100 mumole/l. It is concluded from the data obtained that pentamidine and related diamidines in the 2 assays tested show no mutagenic or genotoxic effects, in spite of their tight binding to DNA.

Effects of naftidrofuryl on the indocyanine green clearance and cardiovascular function of rats with liver cirrhosis.

The influence of intrahepatically infused naftidrofuryl (Dusodril) on indocyanine green (ICG) clearance was measured in healthy rats and rats with liver cirrhosis. In control rats naftidrofuryl reduced the ICG clearance from 0.043 to 0.029 mumol/min/g liver. Plasma half-life of ICG was prolonged significantly from 3.07 to 4.25 min. This was probably due to a depression of the cardiovascular function (blood pressure, heart-rate). In cirrhotic rats, the removal rate of ICG (0.050 mumol/min/g liver) was significantly lower and its half-life (7.61 min) longer than in normal rats. Probably the extraction rate for ICG was so low that naftidrofuryl did not further impair the reduced ICG clearance and half-life inspite of reduced diastolic blood pressure and heart rate.

Comparative evaluation of hepatotoxic side effects of various new trypanocidal diamidines in rat hepatocytes and mice.

A series of new trypanocidal diamidines was tested for their hepatotoxic potential. At a concentration of 0.5 mmol/l almost all agents tested produced leakage of lactate dehydrogenase and reduced viability of isolated rat hepatocytes. In mice serum sorbitol dehydrogenase was raised by a single i.p. dose of 65.5 mumol/kg. Only two agents, compounds 261/115 and 313/40, were free of these effects at this dose or concentration.

Acinar distribution of glutathione-dependent detoxifying enzymes. Low glutathione peroxidase activity in perivenous hepatocytes.

The acinar distribution of glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G-6-PDH) was examined by analyzing periportal (p.p.) and perivenous (p.v.) rat hepatocytes selectively isolated by the digitonin-collagenase perfusion. The cytosolic GST activity was higher in p.v. cells, but the microsomal GST and cytosolic GR were found to be evenly distributed in the acinus. In contrast, the activity of both the Se-dependent GPx and the microsomal (Se-independent) GPx, as well as G-6-PDH, was much lower in the p.v. than in the p.p. cells. The heterogeneous distribution of GST, GPx and G-6-PDH was confirmed by analyzing liver perfusion effluents collected after ante- or retrograde digitonin infusion. The relatively low activities of GPx and G-6-PDH in the p.v. cells could partly explain the susceptibility of this region to chemical injury.

Effects of a small dose of ethanol and calcium carbimide-induced acetaldehyde intoxication on human platelet aggregation, associated thromboxane formation and urinary excretion of 2,3-dinor-6-keto prostaglandin F1 alpha.

We studied ADP-induced platelet aggregation, associated thromboxane B2 (TXB2) formation, urinary excretion of the prostacyclin metabolite 2,3-dinor-6-keto prostaglandin F1 alpha (2,3-dinor-6-keto PGF1 alpha) and formation of malondialdehyde in 10 healthy volunteers after ingestion of a small dose of ethanol (0.25 g per kg of body weight) and calcium carbimide (50 mg). Platelet aggregation in platelet-rich plasma (PRP) was suppressed (p less than 0.05) by ethanol, but no change occurred in platelet TXB2 formation. Ingestion of calcium carbimide caused significant elevations in blood acetaldehyde (p less than 0.001) and ethanol (p less than 0.05) levels, but acetaldehyde did not influence platelet aggregability or the aggregation-associated TXB2 formation. However, calcium carbimide per se significantly (p less than 0.05) elevated TXB2 formation. No effects were found on plasma malondialdehyde levels and urinary excretion of 2,3-dinor-6-keto PGF1 alpha. These observations indicate that a small dose of ethanol attenuates platelet aggregation without any significant effect on aggregation-associated TXB2 formation. By contrast, ingestion of calcium carbimide per se may enhance TXB2 formation.

Glutathione metabolism in isolated rat hepatocytes: acinar heterogeneity of detoxifying enzymes and effects of ethanol.

Periportal and perivenous hepatocytes were isolated by the digitonin-collagenase perfusion technique. The activity of the cytosolic glutathione S-transferase was higher in perivenous cells, but the cytosolic glutathione reductase and the microsomal glutathione S-transferase activities were evenly distributed. In contrast, both the Se-dependent and the microsomal Se-independent glutathione peroxidase activity and the glucose-6-phosphate dehydrogenase activity was much lower in perivenous hepatocytes, suggesting that these cells have a lowered detoxification capacity, which may contribute to their greater susceptibility to damage by xenobiotics. The mechanism of the ethanol-induced GSH depletion in vivo was studied by incubating conventionally isolated hepatocytes. In the absence of glutathione precursors, ethanol (80 mM) did not influence the GSH content, despite accumulation of acetaldehyde (10-100 MicroM). L-Methionine or L-cysteine stimulated GSH replenishment to in vivo rates. Ethanol oxidation resulted in acetaldehyde accumulation, but did not inhibit GSH replenishment from L-methionine and even stimulated that from L-cysteine. This seems to exclude conjugation of GSH with acetaldehyde as a mechanism by which ethanol suppresses GSH levels in vivo.

Changes in serum transaminases, SDH and liver morphology after treatment with trypanocidal diamidines in mice.

The effects of 2 recently developed trypanocidal agents, Diamidinophenylindol (DAPI) and Diimidazolinophenylindol (DIPI) at doses of 10-30 micrograms/g intraperitoneally (i.p.) on serum GOT, GPT and sorbitol dehydrogenase (SDH) levels and on liver morphology have been investigated in mice. Pentamidine served as reference drug. Both agents caused dose-dependent increases in serum transaminases and SDH, and discrete morphological changes of the liver, e.g., fatty degenerations, azinoperipheral vacuolisation and alterations of the nuclei, at least at the higher dosage.

Effect of ethanol on the microsomal glutathione S-transferase activity in glutathione-depleted rat liver.

Depletion of hepatic glutathione in male rats by starvation caused a significant increase in microsomal glutathione S-transferase activity, which was not affected by acute ethanol pretreatment. An additional depletion in fasted rats by diethylmaleate (0.5 g/kg) caused a further increase in the enzyme activity, but this increase was delayed in ethanol intoxicated rats. Although ethanol caused a small increase in hepatic microsomal lipid peroxidation in control animals, this effect of ethanol was not observed in diethylmaleate treated rats and thus was apparently not responsible for the delay in enzyme activation. It is suggested that the activation of microsomal glutathione S-transferase activity towards 1-chloro-2,4-dinitrobenzene in glutathione-depleted rat liver may be produced by changes in thiol/disulfid ratio and/or some reactive oxygen species.