Einleitung: Es besteht eine Assoziation zwischen der R122H-Mutation des humanen kationischen Trypsinogens (hPRSS1) und hereditärer Pankreatitis. Um die Pathogenese dieser Erkrankung näher zu untersuchen, generierten wir eine R122H_hPRSS1-transgene Maus. Diese Mäuse zeigten im 8. Lebensmonat einen milden Phänotyp, der durch experimentelle Caerulein-Pankreatitis etwas verstärkt werden konnte (1).

Expression of carbamoylphosphate synthetase I (CPSI; EC 6.3.4.16) was examined immunohistochemically in normal development of the mouse liver, and in testicular transplants of fetal liver fragments. CPSI started to be expressed in all hepatocytes around 15 days of gestation, and became heterogeneous (i.e. absent from pericentral hepatocytes) around 2 weeks after birth. Most hepatocytes in fetal liver fragments placed for 2 months under the testicular capsule expressed this enzyme except for the pericentral ones, most of which were positively stained with anti-glutamine synthetase (GS; EC 6.3.1.2) antiserum. This distribution resembled that in the adult liver. The steep change in CPSI immunostaining in liver lobules suggests that the microenvironment tightly connected to the central veins plays an important role in the suppression of CPSI expression in the pericentral hepatocytes. Some pericentral hepatocytes were also negative for both enzymes. Thus, control mechanisms of CPSI expression may be different from those of GS expression in pericentral hepatocytes.

Cocultivation of primary pig hepatocytes with RL-ET-14 cells, an endothelial-like cell line resulted in spontaneous induction of glutamine synthetase (GS)-activity in the hepatocytes by more than 10-fold within 120 h to 144 h. Hepatocyte-specific induction was confirmed by immunocytochemistry. Addition of trijodothyronine (T3) to the culture medium inhibited the induction in a concentration dependent manner. No comparable influence of T3was seen with pure cultures of either cell type suggesting that only the spontaneous induction was affected. Other hormones such as glucagon, insulin, growth hormone, epinephrine and testosterone did not interfere with the induction. Addition of several protein kinase-inhibitors such as staurosporine and genistein were without influence. However, a strong inhibition was found after addition of okadaic acid in nanomolar concentrations indicating an involvement of protein-phosphatase 2A in the induction process.

The dose responses for several effects of low-level limited exposures to 2-acetylaminofluorene (AAF) in the livers of male Fischer 344 rats were measured and a subsequent phenobarbital tumor promotion regimen was used to manifest initiation of carcinogenesis. Three doses over a 10-fold range yielding cumulative total exposures of 0.126, 0.42, and 1.26 mmol AAF/kg body weight were achieved by daily intragastric instillation for up to 12 weeks with interim terminations. This was followed by 24 weeks administration of 500 ppm phenobarbital (PB) in the diet to promote liver tumor development. At 12 weeks at the end of AAF administration, all exposures produced adducts in liver DNA, measured by 32P postlabeling, and the level of adducts increased with exposure, except that the high exposure did not produce a dose proportional increase. Measurement of arylsulfotransferase activity, a key enzyme in the metabolic activation of AAF, revealed that in livers from the high exposure animals, the enzyme was inhibited. To assess for toxicity, the centrilobular zone of glutamine synthetase-positive hepatocytes was quantified immunohistochemically at 12 weeks. The area of the zone was reduced in the high exposure group and there was a trend to reduction in relationship to exposure. The two lower exposures to AAF produced no increase in cell proliferation, whereas the high exposure resulted in a marked increase, about 8-fold over controls. Initiation was assessed by induction of hepatocellular altered foci (HAF) that expressed the placental form of glutathione S-transferase. AAF induced HAF in the high exposure group, 9-fold at 8 weeks and 170-fold at 12 weeks compared to controls. In rats maintained on PB for 24 weeks after exposure, the multiplicity of HAF increased in controls and comparably in the low and mid exposure groups, but remained at the about the same high level in the high exposure group. The high exposure produced a substantial incidence of benign neoplasms by 12 weeks, and with promotion by 36 weeks, all rats developed hepatocellular neoplasia. In the mid exposure group, only one adenoma occurred at 36 weeks in 17 rats, while in the low exposure group, no liver tumor occurred in 23 rats. Thus, these findings document nonlinearities for some of the effects of AAF, with supralinear effects at the high exposure for cell proliferation and induction of HAF, and a no-observed-effect level for induction of promotable liver neoplasms at the lowest cumulative exposure of 0.126 mmol/kg, in spite of the formation of DNA adducts. We conclude that the effects of this DNA-reactive hepatocarcinogen leading to initiation exhibit nonlinearities and possible thresholds.

In a previous study, p-dichlorobenzene (pDCB), which is associated with tumorigenicity in male rat kidney and livers of mice of both genders, was found to produce acute increases in cell proliferation in those tissues. To determine whether sustained cell proliferation in the liver in susceptible species correlated with reported carcinogenic effects, we examined the effect of pDCB on cell proliferation in the livers and toxicity to the glutamine synthetase-expressing hepatocyte (GS+) subpopulation of male B6C3F1C3F1 mice and F344 rats. Mice were exposed for up to 4 weeks to 600, the maximally tolerated dose which increased liver tumors, 300 or 150 mg/kg. Rats were exposed to 300, 150 or 75 mg/kg for up to 4 weeks. In mice, the cumulative replicating fraction (CRF) in the livers of the high dose animals was significantly increased 16-fold at 1 week and 4-fold at 4 weeks. The CRF was also increased at 300 mg/kg at 1 week, but this subsided at 4 weeks. No increase was seen in the low dose group. In rats, the CRFs of the livers at 1 week were increased at 300 and 150 mg/kg, but returned to normal at 4 weeks. The size of the hepatic GS+ area was not affected in mice or in rats after 1 week of exposure, but comparable decreases were observed at all exposures at 4 weeks in mice. The data therefore suggest that sustained increases of cell division in the mouse liver may contribute to the increases in liver tumors. The transient increase in rat liver suggests that this is not sufficient to enhance tumor development. The absence of sustained increases of the CRFs at the low dose in mice, which was one-fourth of the hepatocarcinogenic dose, implies the existence of a threshold in pDCB hepatocarcinogenesis.

Silybin glycosides-23-O-beta-glucoside 2b, beta-galactoside 3b, beta-lactoside 4b and beta-maltoside 5b-have been synthesized by different methods (Helferich glycosylation, Lewis acid catalysis), Separation of two silybin diastereoisomers in the form of acetylated monoglycosides has been achieved for the first time, These new silybin glycosides are 4-30 times more water-soluble, and their hepatoprotectivity is increased compared with that of the parent compound silybin 1.

Primary rat hepatocyte cultures exposed to tert-butylhydroperoxide (t-BHP) or cumene hydroperoxide were used to assess the antioxidative and protective potential of water-soluble extracts of artichoke leaves. Both hydroperoxides stimulated the production of malondialdehyde (MDA), particularly when the cells were pretreated with diethylmaleate (DEM) in order to diminish the level of cellular glutathione (GSH). Addition of artichoke extracts did not affect basal MDA production, but prevented the hydroperoxide-induced increase of MDA formation in a concentration-dependent manner when presented simultaneously or prior to the peroxides. The effective concentrations (down to 0.001 mg/ml) were well below the cytotoxic levels of the extracts which started above 1 mg/ml. The protective potential assessed by the LDH leakage assay and the MTT assay closely paralleled the reduction in MDA production and largely prevented hepatocyte necrosis induced by the hydroperoxides. The artichoke extracts did not affect the cellular level of glutathione (GSH), but diminished the loss of total GSH and the cellular leakage of GSSG resulting from exposure to t-BHP. Chlorogenic acid and cynarin accounted for only part of the antioxidative principle of the extracts which was resistant against tryptic digestion, boiling, acidification, and other treatments, but was slightly sensitive to alkalinization. These results demonstrate that artichoke extracts have a marked antioxidative and protective potential. Primary hepatocyte cultures seem suitable for identifying the constituents responsible for these effects and for elucidating their possible mode of action.

The distribution of glutamine synthetase (GS) in a mammalian liver is restricted to a small zone of hepatocytes surrounding the central veins. The determination of the size of the GS+ zone in rats by immunohistochemistry revealed that it differed between rat strains and was larger in males than in females of each strain. Accordingly, the means of the relative mean width (RMW) values that characterize the size of the GS+ zone were 19%, 26%, and 39% lower in females than in males of Sprague-Dawley, Wistar, and Fischer rats, respectively. Upon orchidectomy of male rats, the size of the GS+ zone diminished towards the value found in females, while ovariectomy was without effect. This orchidectomy-induced reduction was reflected in corresponding changes of the RMW values as well as in the number of GS+ cells per pericentral field and was not due to the slightly smaller size of the GS+ hepatocytes in the orchidectomized males. No such sex difference was found in M775 mice. Biochemical GS activity was higher in the male rats than in the female rats and changed correspondingly to the distribution after gonadectomy. In the mice, only the specific activity of GS dropped after orchidectomy. In primary cultures of rat hepatocytes, no influence of testosterone or estrogen on GS activity and cellular distribution was observed, even after stimulation of GS activity with dexamethasone and growth hormone. Both sex hormones, however, were able to affect the activity of glucose-6-phosphate dehydrogenase (G6PD). The observed sex differences in the activity and distribution of GS in rat livers suggest that sex hormones not only modulate the level of this enzyme but are at least partially involved in the determination of the size of the compartment of GS expression. According to the results in the cell cultures, the effects of the sex hormones appear indirect rather than direct.

The exposure-responses for several effects of limited exposures to diethylnitrosamine (DEN) in the livers of male Fischer 344 rats were measured and phenobarbital promotion was used to enhance expression of initiation of carcinogenesis. Five doses ranging from a cumulative total of 0.5 to 4 mmol DEN per kg body weight were given as weekly i.p. injections for 10 weeks. This was followed by 4 weeks recovery, after which the groups were maintained on either a basal diet or 0.05% phenobarbital (PB) to promote liver tumor development. All doses of DEN produced ethylation in liver DNA, which increased with dose. Indicative of toxicity, the centrilobular zone of glutamine synthetase-positive hepatocytes was reduced in relationship to exposure up to a cumulative exposure of 3 mmol/kg. The two lower exposures to DEN produced no increase in cell proliferation, whereas higher exposures resulted in marked increases, approximately 4-fold between 1.0 and 2.0 mmol/kg cumulative. At the end of the recovery period (14 weeks), hepatocellular altered foci (HAF), which expressed the placental form of glutathione S-transferase, were induced by all exposures, with an increase of approximately 4-fold between the exposures of 1.0 and 2.0 mmol/kg being the greatest. In rats maintained on basal diet or PB for 24 weeks after exposure, HAF increased further and with exposures of 2.0 mmol/kg and above, all rats developed hepatocellular carcinomas. With 1.0 mmol/kg, no liver tumor occurred in 12 rats without promotion, whereas in those given PB, two adenomas and two carcinomas were present in 12 rats. At the lowest exposure of 0.5 mmol/ kg, no tumor occurred in rats on basal diet, although HAF increased approximately 7-fold. With PB promotion, only one adenoma developed in 12 rats and HAF increased another 2-fold. Thus, the findings document non-linearity for some of the effects of DEN and a near no-effect level for initiation of promotable liver neoplasms at the lowest exposure in spite of a substantial induction of HAF.

BACKGROUND: Glutamine synthetase is exclusively expressed in pericentral hepatocytes in mammalian liver, but its regulation mechanism is still largely unknown.EXPERIMENTAL DESIGN: Heterogeneous expression of glutamine synthetase was examined in detail during mouse liver development by immunohistochemistry and by in situ hybridization. Heterogeneous expression of this enzyme was also analyzed in immature liver fragments transplanted to an ectopic site where no portal blood flow exists.RESULTS: At 18.5 days of gestation, a random, spotty distribution of low levels of glutamine synthetase mRNA was observed all over the liver parenchyma, but the enzyme protein was not detectable immunohistochemically in the liver at any fetal stage. Glutamine synthetase and its mRNA began to be heterogeneously expressed in pericentral hepatocytes 2 to 3 days after birth, when glycogen accumulation in the liver parenchyma was rather homogeneous. In the early postnatal development, a mosaic distribution of positive and negative hepatocytes with respect to glutamine synthetase protein and mRNA was noted around the central veins. Subsequently, mRNA distribution gradually became continuous, although some hepatocytes still lacked protein, indicating partial regulation at the translational level. When fetal liver fragments that had not yet heterogeneously expressed glutamine synthetase were cultured under the testis capsule of male mice, only pericentral hepatocytes expressed this enzyme after 2 months. However, the distribution of glutamine synthetase protein- and mRNA-positive hepatocytes around the central veins was patchy rather than continuous, as in perinatal livers.CONCLUSIONS: These results support the importance of local interactions of hepatocytes with intrahepatic cell populations and/or structural elements, Furthermore, they demonstrate that the capacity for the positional expression of glutamine synthetase is already established at a fetal age before expression of glutamine synthetase can be detected.

In a two-stage initiation-promotion experiment the hypothesis was investigated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TE), calculated from data of CYP1A induction in hepatocytes in primary culture, or international TCDD equivalents (ITE) are useful for evaluating the tumor-promoting potency of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) and of a defined mixture (M2) of 49 polychlorinated dibenzo-p-dioxins (PCDDs) in comparison with TCDD. Therefore, female Wistar rats were treated with an initiating dose of N-nitrosomorpholine, and subsequently received daily doses of 2, 20 and 200 ng TCDD/kg or equivalent doses of HpCDD or M2, based on TE values. After a promotion phase of 13 weeks, hepatic PCDD levels, CYP1A activity and the relative hepatic volume of adenosinetriphosphatase-negative or glutathione S-transferase P-positive preneoplastic foci were determined. After logarithmic transformation, linear PCDD level-response relationships were obtained for induction of CYP1A activity with TCDD, HpCDD or M2. Based on TE values, inducing potencies of both HpCDD and M2 were over-estimated at higher doses, whereas induction was approximately equivalent at the lowest dose. The best fit of PCDD level-response relationships of relative hepatic volumes of preneoplastic lesions was achieved using a four-parameter logistic model. Significantly different functions were calculated for promotion with TCDD or HpCDD. It is concluded that (i) different PCDD level-response relationships exist for the induction of hepatic CYP1A activity and the promotion of preneoplastic liver foci, and (ii) that TE or ITE factors provide only a rough estimate of the tumor-promoting potency of a PCDD mixture but may overestimate the risk from exposure to higher-chlorinated 2,3,7,8-substituted congeners such as HpCDD.

The early cellular events in liver carcinogenesis were studied in Fischer-344 male rats that either were fed 200 ppm 2-acetylaminofluorene (AAF) for up to 10 wk or were fed the carcinogen for 8 wk followed by maintenance for an additional 24 wk. By 1 wk of exposure, AAF caused a reduction in the number of glutamine synthetase (GS)-positive centrilobular hepatocytes, an increase in DNA synthesizing hepatocytes in the central areas of the hepatic lobules, and a shift from multinucleated to mononucleated hepatocytes, although overt hepatocellular necrosis was not evident. By 3 wk, altered hepatocellular foci characterized by deficiencies in iron storage (IS-) and collagen production and by expression of gamma-glutamyl transferase (GGT(+)) and placental-type glutathione transferase (PGT(+)) activity appeared. Single PGT(+) cells were also found. During continued exposure, foci increased in number, size, and total area with the increases escalating between 8 and 10 wk of exposure. Cessation of AAF exposure at 8 wk resulted in a slight decrease in the number of foci after a further 6 wk of maintenance, but with continued maintenance for another 6 and 12 wk, the number again increased. IS- characterized the majority of foci during carcinogen administration, whereas after cessation of exposure, GGT(+) and PGT(+) foci predominated. None of the foci were positive for GS. After AAF exposure for 10 wk, a few neoplasms developed and greater numbers occurred after maintenance for a further 24 wk of rats exposed for 8 wk. We conclude the following: (a) the low dose of AAF caused subtle alterations in function and proliferation of normal hepatocytes and converted hepatocytes into focus cells; (b) reduction of the GS(+) area is a sensitive indicator of cytotoxicity of AAF; (c) the development of some foci at an early stage depends on a promoting action of AAF, which ceased when the carcinogen was withdrawn, allowing some foci to undergo reversion; (d) a strong linkage exists in expression of IS-, GGT(+), and PGT(+) in foci; (e) the carcinogenic process accelerates in the absence of any indication of increased cytotoxicity by AAF; and (f) under the conditions of this study, no GS(+) foci, adenomas, and carcinomas were found, indicating that no carcinogen-induced expression of GS occurred in these lesions and that GS expression is not linked to other phenotypic abnormalities.

Recent studies of our group have shown that the oxidation of the substituted anthraquinone skeleton is involved in the biotransformation of mitoxantrone. In this report the importance of this process with regard to the mode of action of the drug is investigated. This communication describes a new high performance liquid chromatography separation for mitoxantrone and its metabolites allowing the direct coupling of high performance liquid chromatography to mass spectrometry. Application of this technique to bile of mitoxantrone-treated pigs reveals the formation of several metabolites in addition to the drug-derived compounds found in urine. Seven biliary metabolites are identified as thioether derivatives of mitoxantrone and its side chain oxidation products. Independent synthesis and structural elucidation of 3 thioether conjugates of the drug provides unequivocal evidence that the hydroquinone moiety of mitoxantrone is the site of reaction with glutathione. Furthermore, the formation of the thioether conjugates in HepG2 hepatoma cells and in rat hepatocytes during cell incubations is demonstrated. Inhibition of cytochrome P-450 with metyrapone prevents the formation of the thioether conjugates and leads to a complete loss of the cytotoxicity of mitoxantrone in HepG2 cells and rat hepatocytes up to concentrations of 200 to 400 microM thereby indicating that mitoxantrone has a negligible effect by itself. Rat hepatocytes were found to be more susceptible for the oxidation-induced cytotoxicity than HepG2 cells. These results demonstrate that the acute cytotoxicity of mitoxantrone depends on prior oxidation of its 1,4-dihydroxy-5,8-diaminoanthraquinone moiety.

This study measured the effect of precise doses of 2-acetylaminofluorene (AAF) in inducing DNA damage, functional changes and neoplastic conversion in rat liver. Groups of male F344 rats at 9 weeks of age were exposed to cumulative doses of 0.5 or 2.0 mmol AAF per kg body weight given by gavage daily 5 days per week over an 8-week period and maintained with no further exposure for up to 8 weeks. Administration of AAF resulted in the formation of N-deoxyguanosin-(8-yl)-2-aminofluorene in liver DNA in relationship to dose. In centrilobular hepatocytes the zone of glutamine synthetase-expressing cells was reduced by exposure. By 8 weeks, but not at 4 weeks, the higher of the two doses of AAF provoked an increase in cell proliferation measured by immunohistochemical incorporation of bromodeoxyuridine. Altered hepatocellular foci expressing the placental form of glutathione transferase were induced by the high dose of AAF at 4 weeks, but not at the low dose. At 8 weeks the incidence of foci at the high dose was 79 times that induced by the low dose. These foci were highly proliferative. In animals exposed to AAF for 8 weeks and maintained for 4 weeks with no exposure, DNA adducts decreased by 80% and cell proliferation subsided by 80%, although the glutamine synthetase zone remained diminished. After discontinuation of AAF, the number of foci diminished by 50% and their proliferation subsided by 80% at 4 weeks, indicating a phenotypic reversion of many foci. With this protocol of administration of precise doses of AAF, we have established non-linearity of effects and a lack of correlation between DNA adduct formation and induction of cellular lesions. We suggest that doses in the range of those reported can be used to study the contribution of epigenetic and genotoxic effects in carcinogenesis and to study threshold events.

Neoplastic conversion induced in rat liver by diethylnitrosamine (DEN) was quantified by measuring preneoplastic and neoplastic lesions over a 34 week period in the beginning of which the carcinogen was given at three dose levels and two dose rates for the first 10 weeks, after which animals were maintained for 24 weeks with either no further exposure or were fed phenobarbital (PB) to promote neoplastic development of cells converted by DEN. DEN was injected s.c. in male F344 rats at weekly or biweekly intervals for total doses of 1, 2 or 4 mmol/kg body wt and then the rats were maintained on basal diet alone or diet containing 0.05% PB. At the end of exposure, DEN had produced a dose-related decrease in centrilobular glutamine synthetase-expressing (GS+) hepatocytes which is indicative of mild cytotoxicity. All doses induced foci that were gamma-glutamyltranspeptidase-positive and iron storage-deficient. The multiplicity of foci in the middle dose exceeded that in the low dose by about a factor of two and, in the high dose, was > 10-fold greater. A few GS+ foci were found in the high dose group only. At 34 weeks, neoplasms were present in the middle and high dose groups. Administration of PB after DEN increased the multiplicity of foci in all dose groups, most substantially in the low dose group. The effect of PB on liver neoplasm yield was marginal in the low non-carcinogenic dose, whereas it enhanced the multiplicity in the weakly carcinogenic middle dose by approximately 10-fold. Four principal findings were made: (i) even at the low doses used, a mild cytotoxic response not evidenced by morphological changes in conventional histopathology was manifested in the GS+ centrilobular subpopulation of hepatocytes; (ii) the dose response over a 4-fold dose range of DEN alone and when followed by PB was non-linear; (iii) the precursor role of foci in the evolution of liver neoplasms was evident and was most conspicuous in the case of GS+ foci; and (iv) a high level of foci induction was required for the evolution of neoplasms, even with PB promotion. The finding of non-linearity with increasing doses of DEN raises questions about the assumption that effects of carcinogens at high doses can be quantitatively extrapolated to low doses.

Exposure of primary rat hepatocytes and human HepG2 cells to water-soluble garlic extracts resulted in the concentration-dependent inhibition of cholesterol biosynthesis at several different enzymatic steps. At low concentrations, sterol biosynthesis from [14C]acetate was decreased in rat hepatocytes by 23% with an IC50 (half-maximal inhibition) value of 90μg/mL and in HepG2 cells by 28% with an IC50 value of 35 μg/mL. This inhibition was exerted at the level of hydroxymethylglutaryl-COA reductase (MHG-CoA reductase) as indicated by direct enzymatic measurements and the absence of inhibition if [14C]mevalonate was used as a precursor. At high concentrations (above 0.5 mg/mL), inhibition of cholesterol biosynthesis was not only seen at an early step where it increased considerably with dose, but also at later steps resulting in the accumulation of the precursors lanosterol and 7-dehydrocholesterol. No desmosterol was formed which, however, was a major precursor accumulating in the presence of triparanol. Thus, the accumulation of sterol precursors seem to be of less therapeutic significance during consumption of garlic, because it requires concentrations one or two orders of magnitude above those affecting HMG-CoA reductase. Alliin, the main sulfur-containing compound of garlic, was without effect itself. If converted to allicin, it resulted in similar changes of the sterol pattern. This suggested that the latter compound might contribute to the inhibition at the late steps. In contrast, nicotinic acid and particularly adenosine caused moderate inhibition of HMG-CoA reductase activity and of cholesterol biosynthesis suggesting that these compounds participate, at least in part, in the early inhibition of sterol synthesis by garlic extracts.