Comparison of DNA adduct levels associated with oxidative stress in human pancreas

DNA adducts associated with oxidative stress are believed to involve the formation of endogenous reactive species generated by oxidative damage and lipid peroxidation. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been carried out. In this study, we isolated DNA from the pancreas of 15 smokers and 15 non-smokers, and measured the levels of 1,N⁶-etheno(2′-deoxy)guanosine (edA), 3,N⁴-etheno(2′-deoxy)cytidine (edC), 8-oxo-2′-deoxyguanosine (8-oxo-dG), and pyrimido[1,2-]purin-10(3H)-one (m₁G). Using the same DNA, the glutathione S-transferase (GST) M1, GSTT1, and NAD(P)H quinone reductase-1 (NQO₁) genotypes were determined in order to assess the role of their gene products in modulating adduct levels through their involvement in detoxification of lipid peroxidation products and redox cycling, respectively. The highest adduct levels observed were for m₁G, followed by 8-oxo-dG, edA, and edC, but there were no differences in adduct levels between smokers and non-smokers and no correlation with the age, sex or body mass index of the subject. Moreover, there was no correlation in adduct levels between edA and eC, or between edA or edC and m₁G or 8-oxo-dG. However, there was a significant correlation (r=0.76; p<0.01) between the levels of 8-oxo-dG and m₁G in human pancreas DNA. Neither GSTM1 nor NQO₁ genotypes were associated with differences in any of the adduct levels. Although the sample set was limited, the data suggest that endogenous DNA adduct formation in human pancreas is not clearly derived from cigarette smoking or from (NQO₁)-mediated redox cycling. Further, it appears that neither GSTM1 nor GSTT1 appreciably protects against endogenous adduct formation. Together with the lack of correlation between m₁G and edA or edC, these data indicate that the malondialdehyde derived from lipid peroxidation may not contribute significantly to m₁G adduct formation. On the other hand, the apparent correlation between m₁G and 8-oxo-dG and their comparable high levels are consistent with the hypothesis that m₁G is formed primarily by reaction of DNA with a base propenal, which, like 8-oxo-dG, is thought to be derived from hydroxyl radical attack on the DNA.     

Carbendazim-induced abnormal development of the acrosome during early phases of spermiogenesis in the rat testis

Effects of a single, high dose of orally administered carbendazim (100 mg/kg) on acrosome formation in the early phases of spermiogenesis were examined by electron microscopy and immunocytochemistry up to day 7.5 post-treatment. No obvious abnormality of acrosome development was noted in the Golgi phase spermatids on day 1.5 post-treatment. On day 3, step 1 spermatids were seen in stage III seminiferous tubules. In stage V tubules at this post-treatment interval, direct connections between the trans-side saccules of the Golgi stacks and the outer acrosomic membranes were observed in step 5 spermatids. Similar direct connections between these two organelles were also observed in the advanced round spermatids in later stages at days 4.5 and 7.5. On day 4.5, step 1 and 3 spermatids were seen in stage V tubules. On day 7.5, round spermatids with various abnormalities of acrosome development were observed in stage VII tubules, in addition to the discontinuous and granular acrosomes reported previously. These features were not observed in testes of control animals. In the immunocytochemical analysis using an antibody mMN7 that recognizes a protein delivered from the Golgi apparatus to the acrosome, spermatids exposed to carbendazim showed various abnormal immunostaining patterns in the acrosomes. On the other hand, strong immunoreactivity was observed in the Golgi saccules connecting to the acrosomes. These results suggest that in testis treated with carbendazim acrosome development is impaired during the early phases of spermiogenesis, and material supply from the Golgi apparatus to the acrosome is perturbed, which is a possible cause of the abnormal development. Received: 31 March 1998 / Accepted: 28 May 1998     

The effect of processing of Chlorellavulgaris: K-5 on in vitro and in vivo digestibility in rats

Two experiments were done to clarify whether or not cell rupture is necessary to improve the digestibility of major components of Chlorella vulgaris: K-5. Chlorella was treated with or without high pressure homogenization (1 × 10⁸ N/m² at less than −20°C) after a heating process (100-120°C). Chlorella (air-dry matter) contained 934 g dry matter and 244 g essential amino acids (total)/kg. Chemical composition was hardly altered irrespective of the treatment. In the first experiment, pepsin digestibility of chlorella protein was determined in vitro. The cell rupture by high pressure homogenization caused a small but significant improvement in pepsin digestibility of chlorella protein compared with the control. In the second experiment, total tract apparent digestibilities of chlorella were determined in the rat. Digestibility of chlorella protein was significantly enhanced by high pressure homogenization, but the difference (88.6% vs. 87.4%, P < 0.01) due to treatment was small and similar to that observed in the in vitro experiment. These results suggested that Chlorella strain vulgaris: K-5 may be an efficient protein source even without cell rupture.     

Elektronenmikroskopische Untersuchungen an Zellen des Ductus thoracicus der Ratte nach Osmium-Zink-Jodid (OZI) Imprägnation

Zusammenfassung Die Lymphozyten des Ductus thoracicus von unbehandelten weißen Ratten und von Ratten nach Sensibilisierung mit Meerrettichperoxydase wurden nach einer zweistündigen Vorfixierung in 2,5% Glutaraldehyd in dem Osmium-Zink-Jodid-Gemisch nach Maillet (1959) inkubiert und elektronemikroskopisch untersucht. Es zeigte sich, daß bei den unbehandelten Tieren nur wenige Lymphozyten keine Reaktion im Kernspalt aufwiesen — ER und Golgi waren nicht ausgebildet —, während die Mehrzahl der Zellen eine starke OZI-Imprägnation des Kernspalts, des ER, des Golgifeldes und der Mitochondrienmatrix zeigte. Die Membransysteme und Mitochondrien der Plasmazellen, die in der Ductuslymphe nach Sensibilisierung auftraten, waren ebenfalls stark OZI-positiv. Auf Grund dieser Befunde halten wires für sehr wahrscheinlich, daß das OZI-Gemisch mit bestimmten Proteinen reagiert, auch mit Antikörpern. Bei einer Erhöhung des pH-Werte auf 6,5 findet in den genannten Systemen keine Reaktion mehr statt.

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Electron microscopic studies of ductus thoracicus cells of the rat after osmium-zinc-jodide (OZI) impregnation

Summary Ductus thoracicus lymphocytes from untreated white rats and from rats after sensibilisation with horseradish peroxydase were incubated in Maillet's (1959) osmiumzinc-jodide mixture after prefixation in 2.5% glutaraldehyd, and examined electron microscopically. It is demonstrated that among the untreated lymphocytes only few cells show no reaction product in the perinuclear space—ER and Golgi A. are not to be observed—while the majority exhibits strong reaction in the nuclear envelope, the ER, the Golgi field and the matrix of the mitochondria. All plasma cells, present in the thoracic duct lymph after sensibilisation with horseradish peroxidase show also a strong OZI-reaction in their cisternal elements and in the mitochondria. According to these findings there is strong evidence that the OZI-solution reacts with certain proteins also with antibodies. Setting the pH of the OZI-solution to 6.5 no more reaction is found in the formentioned systems.

     

Intercellular bridges and division patterns of rat spermatogonia

Summary The pattern of intercellular cytoplasmic bridges between rat spermatogonia and between spermatocytes is illustrated from electron microscopy of serial sections. Clones, or syncytia, containing as many as 22 connected spermatogonia and as many as 74 connected spermatocytes were observed. The absence of closed rings of cells agrees with the observation that intercellular bridges are the result of incomplete cell division, rather than cell fusion. The bridges thus are a record of spermatogonial divisions within a clone. In early spermatogonial generations there is a predominantly linear arrangement. The groups of spermatocytes have more side branches. From the presence of synaptonemal complexes it is concluded that the connected spermatocytes of a given clone are in about the same developmental stage. The pattern of intercellular bridges indicates, however, that not all nuclei in a clone undergo mitosis in the same cycle. The connected cells of a clone are therefore not all of the same generation. From unconnected bridges it is assumed that new clones originate from single cells or groups of spermatogonia which separate from an existing clone.Our thanks to Dr. Y. Clermont of McGill University, Montreal, and to Dr. I. B. Fritz and Dr. W. R. Bruce of the University of Toronto, who discussed our results and their interpretation with us. Financial support for this research was provided by the National Research Council of Canada and by the Canadian Medical Research Council (ME3333). A part of the electron microscopy was done at the International Embryological Laboratory in Utrecht, The Netherlands. The use of the facilities there is gratefully acknowledged.     

The development of sites of metaplastic change in regenerating tendon

Summary The development of cartilage and bone in the regenerating segment of the tendon of Achilles following transection has been studied with regeneration taking place in situ, and also following transplantation to a subcutaneous site. Prior to transplantation regeneration was allowed to proceed in situ for various periods of time.It was observed that cartilage and bone develop from the cells of certain pre-cartilaginous areas which represent a metaplasia from fibroblasts. Transplantation to the subcutaneous site at a stage of regeneration when pre-cartilaginous or cartilaginous foci are present leads to the eventual development of bone in the transplant. Transplants made prior to the development of pre-cartilage or cartilage do not show bony metaplasia.It is concluded that the tension of muscle pull is a factor stimulating the metaplastic transformation of fibroblasts to chondroblasts, but once this transformation has occurred the progression to bony metaplasia continues, independently of tension.Supported by a grant from the Medical Research Council of Canada.     

Morphometrische Befunde zum submikroskopischen Aufbau des Sehnerven der Ratte

Zusammenfassung An 10 normalen Sehnerven (Länge: 10 mm; Durchmesser: 0,5 mm) erwachsener Albino-Ratten wurden morphometrische licht- und elektronenmikroskopische Untersuchungen mit der Treffermethode an 3 Stellen durchgeführt: 1 mm vor dem Bulbus oculi (Meßort A); Mitte zwischen Bulbus und Chiasma (Meßort B); 1 mm vor dem Chiasma opticum (Meßort C). Die statistisch signifikanten Ergebnisse der Untersuchung sind: 1. die Gesamtzahl der markhaltigen Fasern im Sehnerven der erwachsenen Ratte beträgt in elektronenmikroskopischen Aufnahmen 107152±6780. 2. Der relative Volumenanteil der Markfasern (Axone und Markscheiden) nimmt von A über B nach C um 13,3 Vol.- % zu, der relative Volumenanteil des Interstitiums (Glia, Gefäßmesenchym und extracellulärer Raum) um den gleichen Betrag ab. 3. Mit der Volumenabnahme des Interstitiums korreliert eine Abnahme der Gliakernzahl pro mm³ von A über B nach C um 46,2% und eine Abnahme der Dichte des Capillarnetzes gemessen an der Zahl der Endothel- und Pericytenkerne. 4. Die Größe der Querschnittsflächen durch den Sehnerven ist am Meßort B (Mittelabschnitt) kleiner als bei A und C. 5. Die Berechnung der absoluten Volumina aus den relativen Volumenwerten und der Querschnittsflächengröße ergibt, daß das Volumen des Interstitiums zwischen A und B um 34,8% abnimmt, während das Markfaservolumen hier fast unverändert bleibt. Dagegen nimmt zwischen B und C das Markfaservolumen um 11,8% zu, während das Volumen des Interstitiums weiter, aber nur geringfügig abnimmt. Die Ergebnisse zeigen, daß die Umfang. zunahme des Sehnerven vom Mittelabschnitt (Durchtritt durch den Canalis opticus) gegen beide Endabschnitte durch Volumenvermehrung jeweils verschiedener Gewebskomponenten bedingt ist.

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Stereological study on the submicroscopic structure of the rat optic nerve

Summary The normal optic nerves of ten adult albino rats were studied using stereological methods of cell counting in light microscopic preparations and point-hit counting on electron micrographs. The observations were confined to three locations of the nerve: one mm from the eyeball (point A), one mm from the optic chiasm (point C) and midway between these sites corresponding to the canalis opticus (point B). The statistically significant results are: 1. the total number of myelinated nerve fibres in the optic nerve is 107152±6780. 2. The volume of myelinated nerve fibres (axons plus myelin sheaths) increases by 13.3% from point A to C, whereas the volume of interstitium (glial cells, capillaries and extracellular space) decreases concomitantly. 3. The decrease in interstitial tissue corresponds to a 46.2% decrease in glial cells between A and C and of capillaries indicated by the numbers of endothelial cells and pericytes. 4. The surface area of cross sections of the optic nerve at point B is smaller than at point A and point C. 5. The absolute volume of the two structural components in the optic nerve, calculated from percentage volume and cross section surfaces, showed that the volume of interstitial tissue decreases from the eyeball to the intermediate portion by 34.8% whereas the volume of myelinated fibres remains nearly constant. From the intermediate portion to the optic chiasm the volume of myelinated fibres increases by 11.8% whereas the interstitial volume further decreases slightly. Thus the increasing diameter of the optic nerve from the middle part to both ends is caused by increases in volume of different tissue constituents.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Electron microscope study of the myofibril partial disintegration and recovery in the mitotically dividing cardiac muscle cells

Summary The ultrastructure of the myocyte at all phases of mitosis as well as of early postmitotic cells has been studied in the myocardia of 14- and 18-day rat embryos and 5- and 7-day old rats. The myofibrils remain unchanged up to the late prophase. In prometaphase the majority of Z-disks in embryo myocyte myofibrils and considerable part of these disks in myofibrils of suckling rats are drastically disintegrated.This is followed by a progressive isolation and scattering of the myofilament bundles and of the whole sarcomeres during the subsequent phases of mitosis. Thick myofilaments seem to be unchanged but thin ones become frequently poorly outlined (mainly in embryos). The sarcoplasmic reticulum, including its typically differentiated subsarcolemmal cisternae, exhibits relatively few changes during mitosis. In the early postmitotic period there is a gradual restoration of contrast-rich Z-bands, interconnecting the previously isolated sarcomeres. Patterns of this process have much in common with early stages of myofibrillogenesis (appearance of subsarcolemmal Z-bodies, formation of skeins of thin filaments etc.). The cleavage furrow formation is either absent or considerably retarded up to the postmitotic period.Behaviour of some other organelles during myocyte mitosis has been described. Possible mechanisms and significance of the observed phenomena are discussed.The author is greatly indebted to the late Professor L. N. Zhinkin for his interest in this work. The valuable technical collaboration of N. V. Seina as well as the assistance of V. M. Semenov in operating the electron microscope are gratefully acknowledged.     

Die Feinstruktur des ektopischen Schmelzorgans bei beschleunigter Eruption des Rattenschneidezahnes

Zusammenfassung Durch regelmäßig wiederholtes mechanisches Kürzen des rechten unteren Rattenschneidezahnes wurde eine beschleunigte Eruption hervorgerufen. Nach 15–17 Tagen trat eine Störung der Schmelzentwicklung in Form einer Produktion von kreideweißem statt normal pigmentiertem Schmelz und ein rotbräunliches Häutchen im Bereich des gingivalen Epithelverschlusses in Erscheinung. Dieses Häutchen wurde nach intravitaler Fixierung mit Glutaraldehyd abpräpariert und licht- und elektronenmikroskopisch untersucht.Das Häutchen besteht aus mehreren übereinander gelagerten Zellschichten: aus Ameloblasten, einem typischen Papillarorgan, einer Bindegewebsschicht und aus flachen Deckzellen. Die Ameloblasten im Bereich des Epithelverschlusses befinden sich im Stadium der frühen, die Ameloblasten des aus dem Epithelverschluß hervorwachsenden Häutchens dagegen im Stadium der späten Maturation. Die Ultrastruktur des gesamten Papillarorgans läßt auf eine intensive metabolische Funktion und auf Ionentransport schließen. Pigmentgranula, die bei normaler Eruption im Maturationsstadium sowohl im Papillarorgan als auch in den Ameloblasten vorkommen, wurden nur im Bereiche des Epithelverschlusses in vaskulären Elementen und in perivaskulären Bindegewebszellen gefunden. Die Oberfläche des Häutchens wird von einer schmalen Schicht flacher, organellenarmer Zellen gebildet.Das bei beschleunigter Eruption aus dem Epithelansatz hervorwachsende Häutchen ist ein ektopisches Schmelzorgan, dessen rotbräunliche Färbung von der reichen Gefäßversorgung herrührt.

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The fine structure of the ectopic enamel organ in accelerated eruption of the rat incisor

Summary An accelerated eruption of the right mandibular rat incisor was induced by its repeated, mechanical shortening. Fifteen to seventeen days after beginning of the experiment a disturbance of enamel development occured, manifested by production of white, opaque enamel instead of normally pigmented enamel and by appearance of a redbrownish membrane overlying the enamel. This membrane was intravitally fixed with glutaraldehyde and after dissection processed for histologic and electronmicroscopic investigation.The membrane consists of ameloblasts, a typical papillary organ, a connective tissue layer and of flat surface cells. The ameloblasts near the epithelial attachment are in early maturation state, the ameloblasts of the outgrowing membrane show signs of reduction, typical for late maturation. The papillary organ of the entire membrane showes ultrastructural equivalents of high metabolic activity as well as of fluid transports.Pigment granules, which in normal eruption occur in the maturation state in the papillary cells and in ameloblasts, are absent in these cells of the membrane. They were only seen in perivascular elements and in connective tissue cells near the epithelial attachment. On the oral surface the membrane is covered by a thin layer of flat, degenerated cells poor in organelles. These findings support the concept, that the membrane growing out of the epithelial attachment in accelerated eruption of the rat incisor, is an ectopic enamel organ. The red-brownish colour is not due to pigment accumulation but to its rich vascularization.

     

On the fine structure of the perivascular cells in the neural lobe of rats

Summary The purpose of this study is to investigate the ultrastructural features of perivascular cells as found in the neural lobe of the hypophysis. In particular, an inquiry was made into the nature of the relationship of such cells to neurosecretory fibers and endings. The latter, in fact, are often invaginated within the perivascular cells and enveloped by their processes; furthermore, they often reveal a certain number of empty granules as well as characteristics of degenerative nature. In the course of this study the localization of the perivascular cells has been investigated as well as that of their processes within the extensive interlobular network typical of the hypophysial neural lobe of rodents. Based on the data gathered, the hypothesis is put forward that the perivascular cells play an important role in the turnover of neurosecretory endings, both under physiological and experimental conditions, contributing thereby to the release of post-hypophysial hormones.With the technical assistance of Vincenzo Panetta.