Simultaneous occurrence of a thyromediastinal muscle, a truncus bicaroticobrachialis, and a left superior vena cava.

A case is presented of a combination of anatomical anomalies found in a 67-year-old female cadaver during routine dissection by medical students. They include a thyromediastinal muscle, a truncus bicaroticobrachialis, and a left superior vena cava, with complete absence of the right superior vena cava, but with a normal azygos vein opening into the right atrium at the expected site of entry of the superior vena cava. No associated congenital cardiac malformations were found. Clinical implications include the difficulty of heart catheterization through the subclavian veins and misleading images on CT or MRI scans, where the azygos vein could be mistaken for a right superior vena cava.

Modulation of the peroxisomal gene expression pattern by dehydroepiandrosterone and vitamin D: therapeutic implications.

Peroxisomes are ubiquitous organelles required for several metabolic functions. Their dysfunction is responsible for a group of human inherited disorders. In the search for endogenous factors regulating the peroxisomal compartment in normal liver, we treated female rats with dehydroepiandrosterone (DHEA) and 25-hydroxycholecalciferol for 1 and 6 days. Relative transcription levels of 39 selected genes were evaluated by real-time quantitative RT-PCR analysis. Catalase (peroxisomal marker)-specific activity was assayed in total liver homogenate and peroxisomes were visualized by catalase localization. DHEA induced peroxisome proliferation and raised catalase specific activity. Expression levels of 16 (of which 11 were peroxisomal) genes were altered. Pex 11, acyl-CoA oxidase,l - andd -multifunctional enzyme, thiolase 1, phytanoyl-CoA hydroxylase, 70 kDa peroxisomal membrane protein and very long chain acyl-CoA synthetase were upregulated, three others were downregulated. Vitamin D caused downregulation of six genes. Administration of vitamin D to peroxisomal disorder patients may be contraindicated. The adrenocortical hormone DHEA is a potential natural regulator of the peroxisomal compartment. Its therapeutic use in X-linked adrenoleukodystrophy, some other beta-oxidation defects and classical Refsum should be considered.

Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures.

L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat.

Effects of extracellular matrix on the expression of peroxisomes in primary rat hepatocyte cultures.

BACKGROUND/AIMS: Peroxisomes in wild-type cells vary between tissues and developmental stages. In the liver of some peroxisomal deficiency disorder patients, rare parenchymal cells express normal peroxisomes (mosaics); the mechanism is unknown. Our aim was to find factors regulating peroxisome expression. METHODS: Liver-specific as well as peroxisome characteristics were studied in three types of primary rat hepatocyte cultures. RESULTS: Total glutathione S-transferase activity and albumin secretion both increased in the collagen I sandwich and immobilization gel cultures. In contrast, in monolayers cultured on plastic, total glutathione S-transferase activity decreased and albumin secretion was only 30-40% compared to the collagen cultures. Glycogen rosettes typical of liver parenchymal cells were always abundant. Laminin and collagen IV-producing stellate cells were numerous in the monolayer but almost absent in the sandwich cultures. In 6-day-monolayer cultures, the number of liver-specific peroxisomes had decreased while atypical small or elongated peroxisomes appeared. Immunolabeling density for catalase and three beta-oxidation enzymes was decreased compared to adult rat liver; catalase specific activity in homogenates had dropped to 15% and 4% in the sandwich and monolayer cultures, respectively. In 17-day-sandwich cultures, some peroxisomes showed a very weak catalase reaction; total activity was 5%. Supplementation of the collagen type I cultures with several extracellular matrix factors could not prevent peroxisome dedifferentiation. CONCLUSION: The presence of these extracellular matrix components is not sufficient for normal peroxisome expression. It is suggested that hepatocyte-specific and peroxisomal features are regulated differently. The sandwich preserves hepatocyte differentiation better than the monolayer.

Effect of epidermal growth factor in collagen gel cultures of rat hepatocytes.

Collagen gel cultures of hepatocytes represent a promising in vitro model in pharmaco-toxicology. Epidermal growth factor (EGF) is usually added to the culture medium, although one could question its value in a culture model aiming at maintaining a maximum of differentiated functional capacities. In this study, the effects of EGF (20 ng/ml) on albumin secretion, morphology and pentoxyresorufin O-depentylase (PROD), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities have been examined in both collagen gel sandwich and immobilization gel cultures of adult rat hepatocytes. Transmission electron microscopy did not show an obvious influence of epidermal growth factor (EGF) on the intracellular organization of organelles of the rat hepatocytes. It was found that EGF addition had no effect on albumin secretion in both culture models. On the contrary, the presence of EGF in the culture medium provoked in collagen gel sandwich cultures, after 7 days, significant decreases of 66% and 25% in EROD and PROD activities, respectively. On GST activities, no effect of EGF could be observed in both collagen gel cultures. Removal of EGF from the culture medium seemed to have a positive effect on the maintenance of the phase 1 biotransformation capacity of rat hepatocytes. Its addition should therefore be avoided in collagen gel cultures used in pharmaco-toxicology.

Effect of Extracellular Matrix Composition on the Expression of Glutathione S-transferase Isoenzymes in Organotypical Hepatocyte Cultures.

Collagen gel sandwich and immobilization cultures of hepatocytes, using hydrated collagen type I as extracellular matrix (ECM), have been proposed as long-term in vitro models in pharmaco-toxicology. The in vivo ECM composition in the space of Disse is, however, much more complex. As a differentiated hepatocyte phenotype is thought to be highly dependent on ECM composition and biophysical characteristics, we modulated the ECM to mimic the in vivo situation. Moreover, commercially available collagen type I (Boehringer-Ingelheim) was compared to the one prepared in the laboratory from rat tails. ECM composition had no effect on albumin secretion or hepatocyte morphology in both collagen gel sandwich and immobilization cultures. Total, Alpha and Mu class GST activities in organotypical cultures with a complex or a simple collagen type I ECM were similar. The Pi class GST activity increased as a function of culture time in all culture models. Thus, mimicking the in vivo composition of the ECM did not improve the changes in GST expression that were observed in simple collagen gel cultures. The collagen type I matrix is therefore assumed to confer sufficient protection to help the hepatocytes to maintain their differentiated phenotype to a certain extent. Moreover, we hypothesize that the collagen gel matrix may act as a scaffold to keep newly synthesized ECM components in the proximity of the basolateral surfaces of the hepatocytes.

Maturation of the liver-specific peroxisome versus laminin, collagen IV and integrin expression.

The interaction of cells with extracellular matrix components contributes to their specific differentiation. We studied hepatic peroxisomes and their changing features during embryonic development, and we immunolocalized in the same tissue the extracellular matrix components laminin and collagen IV as well as the integrin receptor subunits alpha 1, alpha 2, beta 1, and beta 4. Rat and human embryonic liver peroxisome expression were studied at the light- and electron-microscopic level by means of localizing catalase-, D-amino acid oxidase- and polyamine oxidase activities and by means of the immunocytochemistry of six peroxisomal proteins. The successive import of catalase and the peroxisomal beta-oxidation enzymes, the late appearance of the other enzymes, and the gradual increase of peroxisomal size and number to adult values occurred as asynchronous events. Although still immature, peroxisomes were recognized at every stage examined and coexisted with laminin and collagen IV in both species. The beta 1 integrin subunit was immunodetected as early as at 12.5 days in rat. It was concluded that these extracellular matrix factors may be important for the differentiation of liver parenchyma from the liverbud stage onwards. However, the stepwise maturation of the liver-specific peroxisome suggests the involvement of many other regulating factors.

Biogenesis of peroxisomes in fetal liver.

Peroxisomes are single membrane-limited cell organelles that are involved in numerous metabolic functions. Peroxisomes do not contain DNA; the matrix and membrane proteins are encoded by the nuclear genome. It is assumed that new peroxisomes are formed by division of existing organelles. The present article gives an overview of microscopic studies and recent unpublished results dealing with peroxisome biogenesis in mammalian fetal liver and presents data on peroxisomes in oocytes. Cytochemical (catalase and D-aminoacid oxidase activity) and immunocytochemical data in rat and human liver (antigens of catalase, the three peroxisomal beta-oxidation enzymes, alanine: glyoxylate aminotransferase, peroxisomal membrane proteins with molecular weights of 42 and 70 kDa) indicate that during embryonic and fetal development the peroxisomal population undergoes a differentiation with respect to the composition of the matrix and to the size and number of the organelles. In the youngest stages, rare and small peroxisomes are present, into which the matrix components are imported in a sequential way. The import seems asynchronous in peroxisomes of the same hepatocyte. The size and number of the peroxisomes increase during liver development. In rat and human liver, no morphological or immunocytochemical evidence for an elaborate network of interconnected peroxisomes ("reticulum") was found. Instead, peroxisomes presented as individual organelles, which occasionally show membrane extensions. The importance of the metabolic functions of peroxisomes in human liver is emphasized by the peroxisomal disorders. In the liver of affected fetuses, the microscopic features associated with the defect can already be recognized; i.e., either catalase containing peroxisomes are absent and catalase is localized in the cytoplasm (in fetuses affected with Zellweger syndrome or with infantile Refsum disease) or peroxisomes are present but they are abnormally enlarged (e.g., a fetus affected with acyl-CoA oxidase deficiency). In the quail ovary, numerous peroxisomes are observed in the oocyte and in the granulosa cells during follicle maturation, but not in the full-grown egg. Thus, the mechanism of peroxisome inheritance remains unresolved.