Glucuronidation of 4-methylumbelliferone and 4-hydroxybiphenyl and in vitro induction of UDP-glucuronosyltransferase 2B12-mRNA in precision-cut rat liver slices

Fresh rat liver slices were used to demonstrate the glucuronidation of the model substrates 4-methylumbelliferone (MU) and 4-hydroxybiphenyl (HB). Both glucuronidation reactions proved to be more stable than cytochrome P450-dependent monooxygenations. After an incubation time of 48 h there was no decrease in MU glucuronidation rate, whereas HB glucuronidation was stable until 24 h, and then decreased by about 50% until 48 h.

The technique of quantitative competitive RT-PCR was used to determine the expression of UDP-glucuronosyltransferase 2B12-mRNA (UGT2B12-mRNA) in precision-cut rat liver slices. Constitutive levels of UGT2B12-mRNA were measurable. Following 24 h culture of rat liver slices in the presence of phenobarbital, the level of UGT2B12-mRNA increased about twofold, which corresponds to the inducibility in vivo. The addition of beta-naphthoflavone had no influence. The results show that precision-cut liver slices are not only suitable for the detection of an in vitro induction of cytochrome P450-mRNAs, which is characterized by high induction factors, but also of poor induction effects, e.g. on UGT2B12-mRNA, provided that the respective mRNA is exactly quantified.     

Intracellular changes in rat hepatocytes after intratracheal administration of highly dispersed silicon dioxide and uridine effects on these changes

Rat hepatocytes were examined under electron microscope at early terms after intratracheal administration of highly dispersed silicon dioxide powder against the background of uridine treatment. Penetration of powder particles into hepatocyte cytoplasm, nuclei, mitochondria, and peroxisomes and development of bacteria in these cells were observed. Uridine reduced the destructive effect of powder on the organelles, increased glycogen content in hepatocytes, and inhibited the formation of capsulated bacterial forms in these cells. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 141, No. 5, pp. 596–600, May, 2006     

In vitro induction of cytochrome P4503A1-mRNA and testosterone hydroxylation in precision-cut liver slices from male and female rats

Cytochrome P450 (CYP) 3A is constitutively highly expressed in the liver. Thus detection of induction might be more difficult than shown for scarcely expressed CYP families. In this paper the suitability of rat liver slices to prove CYP3A inducibility was demonstrated. CYP3A dependent basal testosterone hydroxylation (TH) at positions 15β, 6β and 2β was lower in liver slices from female than male rats, but was more markedly induced by 10⁻⁶ M dexamethasone (DEX) within 24 h (mean induction factors 12.5, 18.3 and 140, respectively, for female slices and 3.7, 2.3 and 3.5, respectively, for male slices). Basal expression of CYP3A1-mRNA was stable in vitro until 24 h and did not differ between male and female rats. In liver slices from male rats this mRNA was induced about 14fold by both DEX and pregnenolone 16-carbonitrile (PCN) within 24 h. In one sample of a female rat a similar range of CYP3A1-mRNA induction was reached by DEX.

Altogether, CYP3A induction can be detected more sensitively in liver slices from female than male rats, if TH rates are used as indicators. With liver slices from male rats CYP3A1-mRNA reacts more sensitively to inducers than TH.     

Bile acid transport and metabolism in rat liver slices

To further characterise precision-cut liver slices from 34- to 40-day-old male rats as an in vitro model for bile acid (BA) metabolism and transport, the effect of the primary BAs cholic (CA, 5 microM) and chenodeoxycholic acid (CDCA, 0.15 and 0.75 microM) as well as of the therapeutically used tauroursodeoxycholic acid (T-UDCA, 5 microM) on BA profiles was investigated. After 4 h incubation in 5 ml Krebs-Henseleit buffer (KHB) 26 individual BAs were determined in slices (50 mg liver/5 ml KHB) and medium by HPLC with postcolumn derivatisation and fluorescence detection. In control incubations, mean total BA concentrations were 5.09 nmol/50 mg liver (101.80 nmol/g liver) in slices and 25.71 nmol/5 ml KHB, among them 72% taurine-(T-), 22% glycine-(G-) conjugated and 6% free BAs in tissue and medium. The main BAs were beta-muricholic (beta-MCA and conjugates) and cholic acids (CA and conjugates) in tissue and medium. The following results were obtained after addition of CDCA, CA, and T-UDCA, respectively, to the KHB. The toxic CDCA was quantitatively converted mainly to T-UDCA and taurohyodeoxycholic (T-HDCA) acid. CA was conjugated in equal shares to T- and G-CA, whereas T-UDCA was enriched in slices and hydroxylated half to T-beta-MCA, which is the main BA in rats. In conclusion, rat liver slices are highly effective not only in uptake, conjugation and excretion of BAs but also in conversion of strong detergent into less toxic BAs.     

Specific activation of the different fibrogenic cells in rat cultured liver slices mimicking in vivo situations

Due to the loss of cell–cell and cell–matrix interactions, cell culture models poorly mimic the in vivo situation. Therefore, we tested the applicability of precision-cut liver slices (PCLS) to study the early activation of the two main liver fibrogenic cell subpopulations: hepatic stellate cells (HSC) and portal fibroblasts (PF). PCLS were treated with thioacetamide or acetaminophen to induce HSC activation. In PCLS culture, both were able to trigger centrolobular lesion and HSC activation as observed in vivo. However, thioacetamide also presented a toxic effect on portal tract cells. In this PCLS model of centrolobular lesion, the antioxidant N-acetylcysteine was able to prevent acetaminophen-induced injury. To induce a specific activation of PF, PCLS were treated with epidermal growth factor or β-oestradiol. As in vivo, epidermal growth factor and β-oestradiol induced bile duct epithelial cell proliferation accompanied by PF activation; however, β-oestradiol also triggers sinusoidal cell proliferation. We demonstrated that treatments usually used in vivo to induce liver fibrosis allow, in cultured PCLS, the specific activation of the two main liver fibrogenic cell subpopulations, making this model very useful to study the mechanisms involved in early fibrogenic cell activation.     

Ultrastructural changes in hepatocytes,sinusoidal endothelial cells and macrophages in hypothermic preservation of the rat liver with University of Wisconsin solution

To identify subtle changes which might lead to liver failure after liver transplantation, rat livers stored at 4° C in University of Wisconsin solution for 8, 16, 24, and 32 h were examined by transmission electron microscopy, scanning electron microscopy, cellular matrix maceration and freeze fracture for ultrastructural analysis. Endothelial cells exhibited aggregation of intramembrane particles (IMPs) at 8 h and produced tiny blebs accompanied by marked development of pits. As deterioration advanced, endothelial cells exposed the perisinusoidal faces of hepatocytes directly to the lumen with destruction of sieve plates. They then degraded with loss of IMPs. Macrophages followed a similar deterioration process to endothelial cells. Membranes of hepatocytes did not demonstrate aggregations of IMPs for 32 h. Rough endoplasmic reticulum (rER) lost ribosomes and smooth ER (sER) increased in amount and dilated in an irregular form. Autophagosomes appeared in the cytoplasm, engulfed cytoplasmic matrix containing intracellular organelles and became autophagic vacuoles. At 32 h bile canaliculi were filled with detached vesicles. This may be one of the causes of preservation related bile duct complications after liver transplantation.     

Ultrastructural changes associated with reversible and irreversible suppression of electrical activity in olfactory cortex slices

Summary The fine structure and electrical activity were studied in thin brain sections prepared from the olfactory cortex of the guinea pig and incubated in vitro in standard and modified conditions. In the standard medium, the potential response was maintained with no marked changes for 4–5 hours and thereafter gradually decreased. The ultrastructure of the tissue was well preserved for the initial 2 hours of incubation. After incubation for 5 hours, many empty spaces were noted. Some dendritic stumps lost fine internal structure, but most of the synapses were apparently normal. Cyanide suppressed the potential response, and caused swelling of the nerve terminals and a decrease in the number of synaptic vesicles. The recovery of the response in the standard medium was not accompanied by a full restoration in the fine structure. If slices were incubated in the absence of glucose and oxygen, with cyanide in glucose-free medium, or at a low temperature, the potential response was irreversibly depressed. In these slices, numerous wide spaces of low electron density were noted which were concluded to have been derived, at least partly, from the swollen dendrites.Dedicated to Professor Toshihiko Tokizane on the occasion of his 60th birthday.     

Insight into the involvement of Kupffer cell-derived mediators in the hepatoprotective effect of glycine upon inflammation: study on rat precision-cut liver slices

Objective and design: To investigate the role of inflammatory mediators in the hepatoprotective effect of glycine against lipopolysaccharide (LPS)-induced liver injury in rats.Material or subjects: Male Wistar rats were used (N = 4 or 5 per group). Precision-cut liver slices (PCLS) were prepared for in vitro studies.Treatment: Glycine (10 mM) and LPS (10 g/ml) were added to the incubation medium of PCLS obtained 3 h after LPS intraperitoneal (i. p.) administration (10 mg/kg) or saline injection to rats. Glycine effects were also investigated in vivo by treating rats with a diet containing glycine (5%) during 3 days.Methods: Tissue injury was assessed by measuring adenosine triphosphate (ATP) and glycogen contents of liver tissue as well as by measuring aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activity in the medium (in vitro) or in the serum (in vivo). Tumor necrosis factor-alpha (TNF-), prostaglandin E₂(PGE₂) and NOx (reflecting nitric oxide production) were measured in the incubation medium or in the serum. Histological detection of both ED-2 and peroxidase activity were used as Kupffer cell markers. Student t test or two-way ANOVA were used for statistic analysis.Results: Glycine added to the culture medium increased both ATP and glycogen contents of PCLS from LPS-treated rats, reduced the production of TNF- and NOx whereas PGE₂ secretion by PCLS increased. In contrast to the in vitro effect of glycine, we observed that a glycine-enriched diet decreased PGE₂ secretion in the serum after LPS challenge.Conclusion: The effect of glycine on LPS-induced mediator secretion is different considering in vitro or in vivo situations. Interestingly, glycine in vitro is able to prevent energy status depletion of PCLS occurring upon inflammation, a phenomenon probably linked to change in inflammatory mediator secretion pattern by hepatic immune cells, namely Kupffer cells.Received 30 March 2004; returned for revision 8 July 2004; accepted by I. Ahnfelt-Rønne 5 November 2004     

Ultrastructural localization of xanthine oxidoreductase activity in isolated rat liver cells

Xanthine oxidoreductase (XOR) can exist in a dehydrogenase form (XD) and an oxidase form (XO). The D-form uses NAD as cofactor and the O-form uses oxygen as second substrate and produces oxygen radicals. Both enzymes have a high affinity for hypoxanthine and xanthine as substrate and produce uric acid, a potent antioxidant. In the present study, XOR activity was demonstrated with the ferricyanide method in permeabilized isolated rat liver cells at the electron microscopical level. Moreover, ultrastructural localization of XO activity in these cells was studied with the cerium salt method. Activity of both XOR and XO was found in matrix and core of peroxisomes of rat liver parenchymal cells. Only XOR activity was present as well in the cytoplasm of rat liver parenchymal cells. In Kupffer cells and sinusoidal endothelial cells, XOR activity was demonstrated in vesicles and occasionally on granular endoplasmic reticulum. XO activity was not found in Kupffer cells and sinusoidal endothelial cells. The presence of uric acid oxidase activity in matrix and core of peroxisomes as was found previously suggests further breakdown of purines to allantoin in peroxisomes. It is suggested that the major function of XOR activity in the cytoplasm of rat liver parenchymal cells and in sinusoidal cells is not the production of oxygen radicals, but rather the production of uric acid which can act as a potent antioxidant.     

Ultrastructural changes in rat lungs after 48 h cold storage with and without reperfusion

Using a left lung orthotopic isograft model in AS strain rats, we have investigated ultrastructural changes in lungs preserved for 48 h at 0 degrees C after a simple flush technique. Lungs were examined after storage alone and after storage followed by up to 1 h reperfusion with blood in vivo. Grafts were flushed with either isotonic saline (NaCl) or hypertonic citrate solution (HCA) alone, or with HCA containing either verapamil (a Ca2+ channel blocker), desferrioxamine (a Fe2+ antagonist), prostacyclin PGI2, nifedipine (a Ca2+ channel blocker) or allopurinol (a xanthine oxidase inhibitor). These agents were also given intravenously to both donor and recipient. Storage in NaCl produced gross cytoplasmic swelling and disruption with widespread nuclear injury. Reperfusion for 1 h resolved cell swelling but some endothelial loss and alveolar capillary wall rupture were seen. HCA with or without additional agents protected against cell swelling but endothelial blebbing and widening of the basement membrane occurred. Reperfusion for 1 h led to recovery of the basement membrane thickness but widespread endothelial loss was observed which was reduced by the addition of verapamil, desferrioxamine, nifedipine or allopurinol to the flush, but not by prostacyclin. Examination of lungs reperfused for shorter periods (5 and 15 min) identified three main types of damage to the vascular endothelium: (1) gross cell swelling, (2) detachment of intact endothelial cells from the underlying basal lamina, and (3) attenuation of cytoplasm due to blebbing. The results suggest that endothelial injury occurring on reperfusion is partly Fe2+ and Ca2(+)-mediated and that reactions catalysed by xanthine oxidase (which include oxygen free radical production) may also be important.     

Scanning electron microscopic study of the changes of the luminal surface of rat thyroid epithelial cells following exposure to cold for 6 to 48 hours (author''s transl)

Zusammenfassung Rasterelektronenmikroskopische Untersuchungen von Schilddrüsenfollikelzellen von Ratten, die 6 bis 48 Stunden einer Temperatur von +4° ausgesetzt gewesen waren, haben gezeigt, dass, im Vergleich mit denen von bei 22° gehaltenen Kontrolltieren, die Zahl der apikalen Mikrovilli zugenommen hat. Diese Ultrastrukturunterschiede — die 48 Studen nach Versuchsbeginn statistisch signifikant sind — stehen in Beziehung mit der durch die Kälteeinwirkung bedingten Aktivitätssteigerung der Schilddrüse.

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Summary Scanning electron microscopic observations of thyroid follicle cells of rats exposed to 4° C for 6 to 48 hours were found to show an increased number of apical microvilli in comparison to those of controls kept at 22° C. These ultrastructural differences — which were statistically significant 48 hours after start of the experiments — are related to stimulation of thyroid gland activity by exposure to cold.

Mit technischer Mitarbeit von Frl. Christa Thommen und Herrn P.-A. Milliquet.     

Ultrastructural changes of extraocular muscles in strabismus patients

ABSTRACT

Strabismus is an ocular disorder characterized by partial or complete inability to keep eye alignment. It represents a very common ocular problem at ophthalmology clinics worldwide. The current study aimed to show the most encountered ultrastructural changes in extraocular muscles (EOMs) collected from patients with different forms of strabismus. Nine specimens of EOMs were collected from five patients during strabismus correction surgery and processed for light and electron microscopy examinations. Histologically, skeletal muscle fibers in normal EOMs appeared tight and normally arranged with clear striations. In strabismic muscles, the fibers appeared disarranged, and atrophied, swollen and disintegrated in some situations. By transmission electron microscopy, normal EOMs were formed of skeletal muscle fibers with intact basal membrane and sarcolemma, tightly aligned myofibrils with well-arranged sarcomeres, Z line and H zone, and normally distributed mitochondria. On the other hand, strabismic EOMs revealed vacuolation and degeneration of myofibrils, accumulation of lipid droplets, subsarcolemmal inclusions and clustering of mitochondria. EOMs obtained from a Down syndrome patient with V-pattern infantile esotropia showed extensive vacuolation and disintegration of myofibrils, and extra- and intracellular deposition of collagen fibers. Interestingly, some skeletal muscle cells exhibited features of autophagic cell death with a trial of engulfing process by neighboring cells. In conclusion, our study traces some characteristic ultrastructural changes in strabismic EOMs, most notably, extensive vacuolation, clustering of mitochondria, degeneration of myofibrils and autophagic changes. These changes might be emphasized as possibly secondary to strabismus.     

Ultrastructural changes in the gracile nucleus of the rat after sciatic nerve transection

Summary Ultrastructural changes in the gracile nucleus of the rat have been examined after peripheral nerve injury. The sciatic nerve of adult rats was transected at mid-thigh level, and after survival times ranging from 1 day to 32 weeks sections from the gracile nucleus were prepared for electron microscopic examination. Unoperated animals served as controls. Atypical profiles were regularly observed in the experimental cases at post-operative survival times from 3 days up to 32 weeks. It was sometimes not possible to classify these as pre-terminal axons or terminals, because synaptic contacts could not be identified. The two most common changes throughout the entire post-operative period were greatly expanded myelinated axons, or unmyelinated profiles containing numerous mitochondria, osmiophilic dense bodies and vacuoles. Atypical profiles were occasionally observed in unoperated control animals. The results clearly show that various types of degenerative changes occur in the gracile nucleus after peripheral nerve injury. These changes differ markedly from previously described transganglionic changes in other systems. It cannot be excluded that some of the changes reflect growth-related reactions, although the typical features of axon regeneration could not be found.     

Toga virus-like particles in acute liver failure attributed to sporadic non-A, non-B hepatitis and recurrence after liver transplantation.

Toga virus-like particles (typically 60-70 nm: enveloped with small surface spikes) were detected in the native hepatectomy specimens in 7 of 18 patients grafted for acute liver failure attributed to sporadic non-A, non-B hepatitis and in 2 patients grafted for fulminant hepatitis attributed to anti-epileptic drug hepatotoxicity. These particles were not detected in the hepatectomies from 12 other patients grafted for other causes of acute liver failure, 12 for various chronic liver diseases, and 2 histologically normal livers. Acute hepatic failure, characterized histologically by severe haemorrhagic necrosis, developed 7 days after grafting in 5 patients, all in the non-A, non-B group with toga virus-like particles in native liver. Similar virus-like particles were detected in all grafts and were in greater abundance than in the native livers. The agent may be novel because pre- and post-grafting sera were negative for antibodies against representative panels of arboviruses and in first and second generation antibody tests for hepatitis C virus.     

Microvascular Changes in Human Gastric Carcinomas with Coagulative Necrosis: An Ultrastructural Study

Ultrastructural findings in three cases of gastric carcinoma with coagulative necrosis are reviewed with special emphasis on microvascular changes. Intratumoral microvasculature revealed more or less stabilized vessels. Some were characterized by a close association between pericytes and endothelial cells, whereas others showed laminated basement membrane, with a loose association between pericytes and endothelial cells. Some mural cells exhibited ultrastructural signs of regressive changes, including lipofuscin granules, swollen mitochondria, and cytoplasmic lucency. These findings are discussed in relationship to a number of recent studies of the microvascular injury caused by hypoxia and reoxygenation, in humans and animals.     

Histopathological changes in Yucatan minipig skin following challenge with sulphur mustard. A sequential study of the first 24 hours following challenge

Sulphur mustard (HD) or 'mustard gas' is a potent vesicant chemical warfare agent whose biological effects in man have been well documented. The histopathological features of the developing cutaneous HD lesion in female Yucatan minipigs up to 24 hours post exposure are reported. Following challenge with HD vapour at a concentration of 12.0 μmol/cm² a sequence of ultrastructural changes to keratinocytes occurred which were initially seen in the stratum basale. Condensation of nuclear heterochromatin and loss of euchromatin was accompanied by cytoplasmic swelling and culminated in focal epidermal necrosis which was evident at 24 h after the challenge.
The melanocyte appeared to be the cell type most sensitive to HD challenge with a loss of cytoplasmic electron density in areas of the cytoplasm immediately surrounding the melanosomes, chromatin condensation, nuclear membrane blebbing with mitochondrial and generalized cytoplasmic swelling. Isolated complete cell necrosis and disruption were noted as early as 2 h following the challenge with generalized necrosis being seen from 12 h onwards. From 12 h onwards, areas of basement membrane degeneration were also observed. These appeared similar in ultrastructure to that observed in other animal model systems but did not progress to the large blisters so typical of the human lesion.
Damage to the upper dermis took the form of an inflammatory response typified by vascular endothelial swelling and vacuolation, dermal oedema and inflammatory cell (mainly neutrophil) infiltration.
There was some evidence of transitory epidermal damage outside the prescribed wound area. This finding may be of clinical importance when surgical treatments are being considered.     

Variations in immunoreactivity of angiotensinogen and cathepsins B and H in rat hepatocytes over 24 hours

To examine variations in immunoreactivity of angiotensinogen and cathepsins B and H in hepatocytes over 24 hr, rat liver was examined immunohistochemically. Immunoreactivity of angiotensinogen and cathepsins B and H in periportal and perivenous hepatocytes varied significantly over 24 hr, when analyzed by an image analyzer. In periportal and perivenous hepatocytes, immunoreactivity of angiotensinogen was highest at 0800 hr and lowest at 2000 hr or 0000 hr, whereas that of cathepsins B and H was maximal at 1600 hr and minimal at 0400 hr or 0800 hr. Proteolytic activities of cathepsins B and H in liver extracts varied in parallel to the variations in immunoreactivity of these enzymes. Localization of angiotensinogen in the liver acinus was inversely correlated to that of cathepsins B and H; angiotensinogen was predominantly localized in periportal hepatocytes, but cathepsins B and H were in perivenous hepatocytes at each time point examined. These results suggest that angiotensinogen in hepatocytes is actively synthesized and secreted early in the light period, whereas proteolytic activities in lysosomes of hepatocytes are augmented late in the light period.     

Optimal oxygen tension conditions for viability and functioning of precision-cut liver slices.

Optimal oxygenation of culture media is important for the successful use of liver slices as an in vitro tool for studying liver function. For this reason the influence of 20, 40, 70 and 95% O2 concentration on the viability and metabolism of liver slices was investigated. The slices were incubated in the roller system at 37 degrees C under continuous gassing for 2, 24 and 48 hrs. Protein, DNA and potassium contents were maintained or even increased over time without influence by O2 concentrations. The albumin secretion of slices incubated at 40-95% O2 did not differ, but was much lower at 20% O2. A slight non-significant decrease in albumin secretion after 24 hrs of cultivation could be observed, whereas a much steeper decline was found in all groups after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation (ECOD) did not differ between the various O2 concentrations, but declined from 2 to 48 hrs of incubation. It can be concluded that O2 concentration of 20% is not sufficient to maintain all cell functions of incubated rat liver slices, wheras 40, 70 and 95% are useful O2 concentrations to retain all parameters investigated.