Helmut Denk

Mechanism of cholestasis: III. Interaction of synthetic detergents with the microsomal cytochrome P-450 dependent biotransformation system in vitro A comparison between the effects of detergents, the effects of bile acids, and the findings in bile duct ligated rats

Abstract Synthetic nonionic and anionic detergents interact with the cytochrome P-450 dependent microsomal biotransformation system in three somewhat overlapping stages depending on dose and time of exposure. In the first stage, the detergents bind to P-450, presumably to a lipoprotein region (binding site I). This is reflected in a spectral change of P-450, the enhancement of the P-450 reductase, and the competitive inhibition of the metabolism of other Type I substrates, like aminopyrine. In the second stage the mixed inhibition of the aminopyrine demethylation without a proportional loss of P-450 suggests a blocking or solubilization of the lipoprotein binding site. In the third stage, degradation of P-450 and solubilization of P-450 reductase reflects the breakdown of the enzyme system. These stages correspond to the effect on microsomes of dihydroxy bile acids in vitro and to some degree of bile duct ligation in vivo . These observations suggest a detergent effect of bile acids as an explanation of the alterations of the endoplasmic reticulum in cholestasis.

Mechanism of Cholestasis: 4. Structural and biochemical changes in the liver and serum in rats after bile duct ligation

To determine whether changes in the function of the hepatocellular microsomal biotransformation system play a role in the genesis of cholestasis or result from it, the biochemical alterations in the biotransformation system and the appearance of the endoplasmic reticulum were compared in rats during the 1st 4 days after bile duct ligation. The time course of the changes in the structure of the hepatocytes was also compared with the results of commonly used hepatic tests. Cholestasis is characterized by decreased metabolism of substrates bound to the lipoprotein of cytochrome P-450 despite an increase in the amount of smooth endoplasmic reticulum. This increase occurs after cholestasis is apparent and, therefore, results from it. The changes in the endoplasmic reticulum with hypertrophy of the smooth form may be adaptive in nature. The high but transient elevation of transaminase activity soon after bile duct ligation was not associated with morphological signs of injury. The later injurious effects of cholestasis are thought to be caused, at least in part, by the accumulation of bile salts, which by their detergent action decrease the function of the hypertrophic smooth endoplasmic reticulum.

Hepatocellular Hyalin in Cholestasis and Cirrhosis: Its Diagnostic Significance

Liver tissue of patients with various stages of alcoholic liver injury, with cholestatic liver diseases and with various types of cirrhosis, were examined for the presence of hyalin, mainly by light microscopy and, in selected instances, also by electron microscopy. Hyalin was detected in alcoholic liver disease and in prolonged cholestasis, mainly in primary biliary cirrhosis. It was not found in diseases in which cholestasis was of shorter duration and was predominantly central in location. Electron microscopically, hyalin was identical in alcoholic hepatitis and in primary biliary cirrhosis, although the pattern of injury of hepatocytic organelles was different in these diseases. A morphologic relation between hyalin and cholestasis or any other hepatocellular abnormalities could not be detected. Hyalin was also found in advanced nonalcoholic cirrhosis, in Wilsons disease, and in Indian childhood cirrhosis, and with the exception of the last, it was always associated with peripheral cholestasis. Centrolobular hyalin in specimens with intact lobular architecture is diagnostic for alcoholic liver injury. Peripheral hyalin in the absence of cirrhosis favors primary biliary cirrhosis over extrahepatic biliary obstruction. Hyalin throughout nodules in advanced cirrhosis does not necessarily indicate alcoholic liver disease, Wilsons disease, or Indian childhood cirrhosis, but also occurs in cryptogenic cirrhosis.

Endometrial stromal sarcomas frequently express epidermal growth factor receptor (EGFR, HER-1): potential basis for a new therapeutic approach.

Endometrial stromal sarcomas are rare malignant mesenchymal uterine tumors. The expressions of different epidermal growth factor receptors such as EGFR (HER-1), HER-2, HER-3, and HER-4 have not yet been examined in these tumors. Twenty-three cases of endometrial sarcomas consisting of 20 low-grade endometrial stromal sarcomas and 3 undifferentiated endometrial sarcomas were examined immunohistochemically for EGFR (HER-1), HER-2, HER-3, and HER-4. EGFR (HER-1) was positive in 17 of 23 (74%) cases. While the three undifferentiated endometrial sarcomas were positive for EGFR, 14 of 20 (70%) low-grade endometrial stromal sarcomas showed positive reactions for EGFR. All examined cases were negative for HER-2, HER-3, and HER-4. This study is the first to show common expression of EGFR (HER-1) in endometrial stromal sarcomas. This finding may provide the basis for a new therapeutic strategy using monoclonal antibodies against EGFR (such as cetuximab) or small molecule inhibitors of EGFR (such as gefitinib) in patients with endometrial sarcomas.

Mechanism of cholestasis. 2. Effect of bile acids on the microsomal electron transfer system in vitro

Abstract 1. 1. Dihydroxy bile acids, which give a Type I binding spectrum with cytochrome P-450, increase the initial rate of P-450 reductase in concentration below 1.0 mM. 2. 2. At 1.0 mM they dissociate the flavoprotein P-450 reductase from P-450, resulting in a concentration-dependent decrease of the initial rate of P-450 reductase and of the amount of enzymatically reducible P-450. 3. 3. Degradation of P-450 by dihydroxy bile acids is concentration and time-dependent. 4. 4. Trihydroxy bile acids do not produce these changes.

Alteration of hepatic microsomal enzymes by griseofulvin treatment of mice

Feeding mice a diet containing 2.5% griseofulvin (GF) for 12 days caused an increase in liver weight to 9 per cent of the body weight with a proportional decrease in microsomal protein/g of liver wet weight. With respect to microsomal protein, the cytochrome P-450 content was 50 per cent and cytochrome b5 was 200 per cent of that in control mouse liver microsomes. The amount of increase in cytochrome b5 was approximately the same as the amount of decrease in cytochrome P-450. and there was no change in the total microsomal heme content. The microsomal content of NADH-cytochrome b5 reductase, as measured by ferricyanide reduction, was unchanged, but in agreement with the elevated cytochrome b5 content, NADH-cytochrome c reductase activity was doubled. While the cytochrome P-450 level was low in microsomes after GF feeding, the NADPH-cytochrome c reductase was significantly elevated. Since this enzyme is generally considered to be rate limiting for many mixed function oxidase reactions, its increase may explain the normal to slightly elevated rates of metabolism in vitro of several type I and type II substrates. Although cytochrome b5, has been suggested as being rate limiting for input of a second electron to cytochrome P-450 linked mixed function oxidations, elevation of cytochrome b5 in the microsomes did not change the extent of NADH-synergism of NADPH-supported aminopyrine demethylation. NADH-supported Δ,10-fatty acid desaturase activity, which requires cytochrome b5, was elevated several-fold after GF feeding. In contrast, NADPH-supported lipid peroxidation showed a slower onset after GF treatment; the NADH-supported reaction, however, was slightly elevated.

Inhibition of NADPH-driven microsomal lipid peroxidation by cytosol factor(S). Effect of a fat-free, high carbohydrate diet

Male Swiss mice were given free access to a fat-free, high carbohydrate diet. The liver cytosol fraction from these mice contained a heat-sensitive factor that markedly inhibited microsomal, ferric pyrophosphate stimulated, NADPH-driven lipid peroxidation. The diet-induced factor was apparently incorporated into the microsomes after 12 days of continuous feeding, since lipid peroxidation by these microsomes was strongly diminished. The factor disappeared from the cytosol after 24 h of fasting and reappeared after refeeding the mice with the fat-free, high carbohydrate diet.

Turnover of hepatic cytochrome P-450 in experimental cholestasis

Abstract In mechanical experimental chllestasis, hypertrophy of smooth microsomal membranes was observed. In contrast to typical induction, the membranes were deficient in cytochrome P-450. The total cytochrome P-450 content of the liver, however, as determined in the liver homogenate remained unchanged. To clarify the mechanism of the development of cytochrome P-450 deficient membranes in cholestasis, the half life of the heme portion of cytochrome P-450, and the initial rate of synthesis of cytochrome P-450 and b 5 hemes were compared in bile duct ligated rats and in control animals after labeling the heme by injection of the precursor δ-[4- 14 C]aminolevulinic acid. The half lives were not significantly different, which eliminates the possibility that selective destruction of cytochrome P-450 has occurred. Depression of cytochromal heme synthesis was not observed. During mechanical cholestasis, the relative cytochrome P-450 deficiency is probably caused by proliferation of components of the endoplasmic reticulum other than the hemoprotein.

Molecular Pathology of Cholestasis

Cholestasis will remain a key problem in liver disease as far as differential diagnosis, prognosis and therapy are concerned until its pathogenesis is resolved. It thus challenges the clinician, both gastroenterologist and surgeon; the morphologist; and the physiologist, each of whom uses his own definition, varying from ‘piling up of biliary substances in the blood’ to ‘visible stagnation of bile in the liver’ to ‘inhibition of hepatocellular bile secretion’ (1).

Keratin 18-deficiency results in steatohepatitis and liver tumors in old mice: A model of steatohepatitis-associated liver carcinogenesis

Backround Steatohepatitis (SH)-associated liver carcinogenesis is an increasingly important issue in clinical medicine. SH is morphologically characterized by steatosis, hepatocyte injury, ballooning, hepatocytic cytoplasmic inclusions termed Mallory-Denk bodies (MDBs), inflammation and fibrosis. Results 17-20-months-old Krt18−/− and Krt18+/− mice in contrast to wt mice spontaneously developed liver lesions closely resembling the morphological spectrum of human SH as well as liver tumors. The pathologic alterations were more pronounced in Krt18−/− than in Krt18+/− mice. The frequency of liver tumors with male predominance was significantly higher in Krt18−/− compared to age-matched Krt18+/− and wt mice. Krt18-deficient tumors in contrast to wt animals displayed SH features and often pleomorphic morphology. aCGH analysis of tumors revealed chromosomal aberrations in Krt18−/− liver tumors, affecting loci of oncogenes and tumor suppressor genes. Materials and Methods Livers of 3-, 6-, 12- and 17-20-months-old aged wild type (wt), Krt18+/− and Krt18−/− (129P2/OlaHsd background) mice were analyzed by light and immunofluorescence microscopy as well as immunohistochemistry. Liver tumors arising in aged mice were analyzed by array comparative genomic hybridization (aCGH). Conclusions Our findings show that K18 deficiency of hepatocytes leads to steatosis, increasing with age, and finally to SH. K18 deficiency and age promote liver tumor development in mice, frequently on the basis of chromosomal instability, resembling human HCC with stemness features.