The gallbladder of Uranoscopus scaber L. (teleost perciform fish) is lined by specialized cholecystocytes.

The gallbladder of Uranoscopus exhibits a mucosal surface layer of simple columnar epithelium composed of specialized cholecystocytes. The apices show storage and mucous secretions, typical microvilli, and very apical projections extending deep into the luminal contents. Many organelles and heterogeneous vesicles of diverse size fill the cytoplasm, including neutral mucins, mitochondria, peroxisomes, lysosomal bodies, and lipid-rich deposits with cholesterol inclusions. The fibromuscular layer shows little blood supply and contains scattered lymph-like walls with minute cholesterol inclusions. The remaining muscular, subserosal, and serosal or adventitial layers of this species do not show any histologic differences to those of other vertebrates. It was unexpected to find cholesterol inclusions in the fatty deposits of the cholecystocytes, similar to those noted in human cholesterolosis and in some forms of hypercholesterolemia, in this teleostean. In addition, aggregations of mitochondria and anomalous mitochondrial morphologies were found that resemble oncocytoma-like changes.

Autoschizis of human ovarian carcinoma cells: scanning electron and light microscopy of a new cell death induced by sodium ascorbate: menadione treatment.

Human ovarian carcinoma (MDAH 2774) cells were treated with sodium ascorbate (VC), menadione (VK3), or a combination of both in a ratio 100:1 for 1h and then examined with scanning electron microscopy (SEM) and light microscopy (LM). Light microscopy data corroborated SEM observations, which demonstrated that death of VC+VK3-treated tumor cells occurred primarily by autoschizis. This type of cell death is characterized by a decrease in cell size, cytoplasmic self-excisions, and nuclear and nucleolar morphologic degradations without the formation of apoptotic bodies. Ultimately, cell death results from karyorrhexis and karyolysis. This study illustrates that plasma membrane damage (branching filopodia, blisters, blebs) results from VC treatment; cytoskeletal damage and self-morsellation are caused by VC, VK3 and VC+VK, treatments. The VC treatment results in a 23% decrease in cell diameter while VK3-treated cells decrease cell diameter by 66%. After 1h of VC+VK3 treatment, a heterogenous cell population is found. This population can be resolved into one population whose diameters are 23% smaller than those of sham-treated cells, and a second population whose diameters are approximately twice those of sham-treated cells. This second population is indicative of doublet formation in which the cells appear to be dividing (an early stage of autoschizic cell death). One half of the doublet contains the cell nucleus while the other half consists of cytoplasm and membrane only. The enucleate portion of this doublet will then be excised. When the types of cell death are enumerated following VC+VK3 treatment, 43% of the cells die by autoschizis, 3% by apoptosis, and 1.9% by oncosis. These results confirm that autoschizis is the principal form of cell death that results from the in vitro treatment of human ovarian carcinoma cells with the vitamin combination.

Potential therapeutic application of the association of vitamins C and K3 in cancer treatment.

The decision of stressed cells to die or to survive is made by integrating signals at different levels through multiple check points. However, initiation and continued progression toward cell death by apoptosis in cancer cells may be blocked by mutation of the tumor suppressor p53 or overexpression of members of the bcl-2 family of proteins. The existence of such mechanisms indicates that cancer cells lose the controls regulating their cell cycle. Therefore, the activation of their programmed cell death appears as a major therapeutic target. Oxidative stress can stimulate growth, trigger apoptosis, or cause necrosis depending upon the dose and the exposure time of the oxidizing agent. A large body of evidence supports the idea that oxidative stress induced by redox cycling of vitamins C and K(3) in association surpasses cancer cellular defense systems and results in cell death. The molecular mechanisms underlying such a process are, however, still unknown. Indeed, several types of cell death may be produced, namely autoschizis, apoptosis and necrosis. Combined vitamin C and K(3) administration in vitro and in vivo produced tumor growth inhibition and increased the life-span of tumor-bearing mice. CK(3)-treatment selectively potentiated tumor chemotherapy, produced sensitization of tumors resistant to some drugs, potentiated cancer radiotherapy and caused inhibition of the development of cancer metastases without inducing toxicity in the host. We propose the association of vitamins C and K(3) as an adjuvant cancer therapy which may be introduced into human cancer therapy without any change in the classical anticancer protocols, and without any supplementary risk for patients.

Autoschizis: another cell death for cancer cells induced by oxidative stress.

Scanning and transmission electron microscopy (SEM and TEM) were employed to characterize the cytotoxic effects of vitamin C (VC), Vitamin K3 (VK3) or a VC:VK3 combination on a human bladder carcinoma cell line (T24) following vitamin treatment. T24 cells exposed to VC alone showed membrane defects. VK3-treated cells show greater damage than VC treated cells because they exhibit membrane defects, cytoskeletal damage, excision of cytoplasm, and a substantial decrease in the number of viable cells. VC: VK3 treatment exacerbates the damages, especially intranuclear and nucleolar and induces an extreme reduction of cell size by cytoplasmic self-excision. Conversely, the nuclear envelope remains intact and the rough endoplasmic reticulum (RER) maintains its integrity until karyorrhexis occurs through a new phenomenon of cell death that we have named "autoschizis". From our morphological studies and previous biochemical reports on the topic, we are able to propose that this autoschizic cell death found is induced by oxidative stress.

Ultrastructural aspects of autoschizis: a new cancer cell death induced by the synergistic action of ascorbate/menadione on human bladder carcinoma cells.

Scanning and transmission electron microscopy were employed to further characterize the cytotoxic effects of a ascorbic acid/menadione (or vitamin C/vitamin K3) combination on a human bladder carcinoma T24 cell line. Following 1-h treatment T24 cells display membrane and mitochondrial defects as well as excision of cytoplasmic fragments that contain no organelles. These continuous self-excisions reduce the cell size. Concomitant, nuclear changes, chromatin disassembly, nucleolar condensation and fragmentation, and decreased nuclear volume lead to cell death via a process similar to karyorrhexis and karyolysis. Because this cell death is achieved through a progressive loss of cytoplasm due to self-morsellation, the authors named this mode of cell death autoschizis (from the Greek autos, self, and schizein, to split, as defined in Scanning. 1998; 20: 564-575). This morphological characterization of autoschizic cell death confirms and extends the authors previous reports and demonstrates that this cell death is distinct from apoptosis.

In vivo reactivation of DNases in implanted human prostate tumors after administration of a vitamin C/K(3) combination.

Human prostate cancer cells (DU145) implanted into nude mice are deficient in DNase activity. After administration of a vitamin C/vitamin K(3) combination, both alkaline DNase (DNase I) and acid DNase (DNase II) activities were detected in cryosections with a histochemical lead nitrate technique. Alkaline DNase activity appeared 1 hr after vitamin administration, decreased slightly until 2 hr, and disappeared by 8 hr after treatment. Acid DNase activity appeared 2 hr after vitamin administration, reached its highest levels between 4 and 8 hr, and maintained its activity 24 hr after treatment. Methyl green staining indicated that DNase expression was accompanied by a decrease in DNA content of the tumor cells. Microscopic examination of 1-microm sections of the tumors indicated that DNase reactivation and the subsequent degradation of DNA induced multiple forms of tumor cell death, including apoptosis and necrosis. The primary form of vitamin-induced tumor cell death was autoschizis, which is characterized by membrane damage and the progressive loss of cytoplasm through a series of self-excisions. These self-excisions typically continue until the perikaryon consists of an apparently intact nucleus surrounded by a thin rim of cytoplasm that contains damaged organelles.

The fibre dimensions of uterine smooth muscle of the rabbit following treatment by female sex steroids.

The effects of female sex hormones on the dimensions of myometrial smooth muscle fibres were studied by using ovariectomized rabbits. After one month of treatment, the fiber dimensions of the outer myometrial layer were measured, using cryostat sections. Calculated smooth muscle fiber volume was found to be in the sequence: control < medroxyprogesterone < estradiol < estradiol + medroxyprogesterone < estradiol alone. The measurements show that medroxyprogesterone-treated uteri contain the narrowest and the longest smooth muscle fibres, while estradiol treatment have the largest cells. This study complements previous observations in showing that medroxyprogesterone alone, or in combination with other modulators, contributes to sustain pregnancy by increasing internal resistance of estradiol-primed myometrial smooth muscle fiber fascicles. Our discussion, based on recent literature, shows that this resistance is ultimately controlled by changes in the myometrium innervation, in the repression of some controlling myofibrillar components, in the expression of specific membrane receptors and ionic channels, and in favoring the switching of molecular connexins in gap junctions, making P paramount in maintaining pregnancy. Moreover, other recent observations have also shown that probably an hcG-like hormone actually control P receptors expression in myometrial smooth muscles.

Human bone marrow fibroblasts infected by cytomegalovirus: ultrastructural observations.

Human cytomegalovirus (HCMV) is capable of infecting human bone marrow fibroblastic stromal cells (HBMF-sc). This infection is important to assess in regard to the pathogenesis of HCMV, particularly in immunocompromised patients. Stromal fibroblastic cells were infected by Towne strain of cytomegalovirus (CMV) in vitro. Several ultrastructural features of uninfected HBMF-sc were also described. The CMV-infected cells exhibited significant mitochondrial enlargement, production of dense bodies by the Golgi apparatus and cytoplasmic accumulation. Ultrastructural aspects of HCMV entry in host cells, capture by endosomes, penetration of genetic material in the nucleoplasm, assembly and formation of nucleocapsids were detected and described. Viral fusion and transit through the nuclear envelope were shown along with envelope proliferations. Trafficking of virions, maturation and completion of their cytoplasmic coating were also illustrated. Fully developed virions, defective virions, other apparently-emptied vesicles, multivesicular bodies as well as cytoplasmic dense bodies were illustrated along arrays of microtubules organized by defective centrosomes and constituted a peculiar structure that we termed 'viral field'. While some viral and dense bodies were carried to adjacent sites of the plasmalemma, in order to be extruded from the infected cells, others were concentrated into black holes--dense heterogenous bodies accumulated at the periphery of viral fields. This study further described the functional aspects of HBMF-sc and summarized the unknown aspects of ultrastructural characteristics of HCMV-infected fibroblastic stromal cells which may serve as harmful reservoir for the replication of virus. In addition, the findings of this study may stimulate further investigations about the basic cell biology and functions of the bone marrow stromal cells and may also generate some interests to bone marrow transplantation medicine as to how HBMF-sc can serve as a reservoir in the pathogenesis of CMV infections.

Ultrastructural aspects of cytomegalovirus-infected fibroblastic stromal cells of human bone marrow.

Human cytomegalovirus (HCMV) and bone marrow interactions are important in the pathogenesis of HCMV infections. Human bone marrow fibroblastic stromal cells (HBFM-sc) were infected by Towne strain of cytomegalovirus (CMV) in cell culture. Several cytostructural features of control bone marrow stromal cells are described and compared with those of CMV-infected cells. Under these experimental conditions, HBFM-sc are cell types that can be successfully infected by CMV in vitro. The CMV-infected cells displayed typical features characteristic of DNA virus infections, such as cellular swelling, intranuclear inclusions, nucleolar condensation and disappearance (at the end stage), nuclear envelope proliferation as redundant folds. Other characteristics of CVM-infected cells include mitochondrial enlargement and vacuolization, cytoplasmic dense bodies associated or not with viral particles, accumulation and extrusion of viral particles and dense bodies. These preliminary observations shed some light on human bone-marrow stromal-cell morphology and function, one of the latter being that of a potentially harmful reservoir for CMV virus.

Ultrastructural nucleolar alterations induced by an ametantrone--poly r(A-U) complex.

The current study has documented changes in the ultrastructure as well as in the intranucleolar distribution of rDNA and rRNA in RT4 (human transitional cell bladder carcinoma) cell nucleoli following a 3-h exposure to toxic doses of 50 microM ametantrone (AMT), 200 microM poly (adenylate-uridylate) (poly r(A-U) or an AMT/poly r(A-U) combination with an AMT/polyribonucleotide ratio of 1:4 and a poly r(A-U) concentration of 200 microM. While the main nucleolar components (fibrillar center (F), dense fibrillar component (D), granular component (G) and interstices (I) can be discerned following all treatments, the nucleoli exhibit: compaction, segregation, a decrease in the number of F, an increase in the size of remaining F, margination of intranucleolar chromatin and retention of intranucleolar pre-rRNA and rRNA. The relative abilities of the test agents to induce nucleolar compaction are AMT/poly r(A-U) > poly r(A-U) > AMT > sham-treated, while the abilities of the test agents to induce the remaining nucleolar changes are AMT/poly r(A-U) > or = AMT > poly r(A-U) > sham-treated cells. Poly r(A-U) and the induced interferon induce nucleolar compaction, while AMT produces nucleolar segregation. These results are consistent with a model in which the poly r(A-U) and/or the AMT inhibit DNA transcription and rRNA processing as well as the release of nascent preribosomes from the nucleolus.

Cancer cell necrosis by autoschizis: synergism of antitumor activity of vitamin C: vitamin K3 on human bladder carcinoma T24 cells.

Scanning and transmission electron microscopy and fluorescence light microscopy were employed to characterize the cytotoxic effects of vitamin C (VitC), vitamin K3 (VitK3) or a VitC:VK3 combination on a human bladder carcinoma cell line (T24) following 1-h and 2-h vitamin treatment. T24 cells exposed to VitC alone exhibited membranous damage (blebs and endoplasmic extrusions, elongated microvilli). VitK3-treated cells displayed greater membrane damage and enucleation than those treated with VitC as well as cytoplasmic defects characteristic of cytoskeletal damage. VitC:VitK3-treated cells showed exaggerated membrane damage and an enucleation process in which the perikarya separate from the main cytoplasmic cell body by self-excision. Self-excisions continued for perikarya which contained an intact nucleus surrounded by damaged organelles. After further excisions of cytoplasm, the nuclei exhibited nucleolar segregation and chromatin decondensation followed by nuclear karryorhexis and karyolysis. This process of cell death induced by oxidative stress was named autoschizis because it showed both apoptotic and necrotic morphologic characteristics.

Ultrastructural nucleolar alterations induced by an ametantrone/polyr(A-U) complex.

In the present study we examined the ultrastructural modifications as well as the precise distribution of DNA and RNA in RT4 cell nucleoli following a 3-h exposure to nontoxic or toxic doses of ametantrone (AMT), poly(adenylate-uridylate) (polyr(A-U), or an AMT/polyr(A-U) combination. While distribution of nucleic acids within the various nucleolar components is not modified following all treatments, the nucleoli exhibit several ultrastructural changes: redistribution of the nucleolar components, decrease in the number of fibrillar centers, and increase in the size of the fibrillar centers. The relative frequencies of the test agents to induce the apparition of nucleoli of compact type are AMT/polyr(A-U) > AMT approximately polyr(A-U) > sham-treated, while the abilities of the test agents to induce the nucleolar segregation are AMT/polyr(A-U) approximately AMT > polyr(A-U) > sham-treated cells. These ultrastructural changes are characteristics of drugs that intercalate into DNA and inhibit rDNA transcription as well as rRNA processing and release of nascent preribosomes from the nucleolus.

Ultrastructural aspects of human cystic duct epithelium as a result of cholelithiasis and cholesterolosis.

Although there is a large body of data on the gallbladder and the importance of the cystic duct in surgical procedures, there is insufficient data regarding the morphology of the human cystic duct. In the present study, transmission electron microscopic (TEM) and scanning electron microscopic (SEM) survey of several surgical and autopsy cystic ducts in cholelithiasis and cholesterolosis is reported. In cholelithiasis, similar to gallbladder epithelium, the cystic duct epithelial cells display minor-to-severe alterations of the epithelial surface accompanied by variable erosion of the epithelium. Areas of intact surface epithelium demonstrate microvilli-covered cells coated by a rich glycocalyx and mucous production. In other areas, apical excrescences are associated with mucus hyperproduction and secretory events. Lipoid bodies are also present in many cells and especially in many of the cells' subliminal apical areas. In cholesterolosis, mucous secretory granules appear dilated, fatty deposits are infrequent, and peculiar intracellular cholesterol deposits can be detected in the apical and subapical region of cells and around condensed mitochondria. Following elective cholecystectomies, predominantly in association with cholelithiasis, eroded areas were detected; therefore, it appears that the action of intraluminal calculi may be a principal causative factor in discrete epithelial erosions of the cystic duct. Intraluminal calculi/ debris, along with the alteration of mucus, cell sloughing, and a decreased pool of bile acids and motility may participate in the gallstone nucleation process. The peculiar cholesterol inclusions may also play a role in that nucleating process.

Cholelithiasis induced in the Syrian hamster: evidence for an intramucinous nucleating process and down regulation of cholesterol 7 alpha-hydroxylase (CYP7) gene by medroxyprogesterone.

This report reviews previously published studies from our laboratory and shows some recent morphological data obtained with scanning and transmission electron microscopy regarding gallstone formation and alteration of the gallbladder epithelium in the Syrian hamster model. Both male and female hamsters were treated with female sex steroids (estradiol alone, estradiol and medroxyprogesterone, medroxyprogesterone alone) during one month. The results show that the Syrian hamster is a good model to study bile changes, gallbladder structure changes, including gallstone formation, and the regulation of cholesterol metabolism at the molecular level. Arguments in favor of this animal model are presented and, during gallstone formation, epithelial cell changes, anionic mucus secretion, and formation of gallbladder luminal deposits can be demonstrated. Recent molecular biology observations related to the effect of female sex steroids on liver cholesterol 7 alpha-hydroxylase (CYP7) gene suggest that progestin alone or primed by estrogen down regulates CYP7 transcription and activity. In addition, progesterone in cell culture systems has been shown to enhance intracellular accumulation of free cholesterol by increasing its uptake and synthesis and by decreasing its esterification by inhibiting the activity of acylcoenzyme A: cholesterol acyltransferase. Non-esterified cholesterol is free to migrate to the extracellular spaces and may contribute to nucleation within the bile. It is suggested that these effects of progesterone on cholesterol metabolism combined with the CYP7 gene down regulation, physical changes in the mucus and the hypomotility of the gallbladder and biliary ducts result in hypersaturation of cholesterol in the bile which favors gallstone formation.

Introduction to the biliary tract, the gallbladder, and gallstones.

This paper serves to introduce a topical section of fifteen invited original research contributions dealing with normal and pathological development of the human biliary tract. This section also includes comparative anatomy of the gallbladder and the cystic duct as well as, the formation of gallstone. This series of reports have used advanced microscopic and ancillary techniques to study adaptative changes in gallbladder epithelial cell changes regarding permeability, renewal, mucous secretion as well as cholesterol uptake and nucleation. Several contributions deal with the blood and lymphatic drainage of the gallbladder. The gallbladder contractility is clarified by recent findings about its innervation, elegantly demonstrated and supported by complementary immunohistochemical and neurophysiological techniques. In vivo models for production of cholelithiasis in the ground squirrel and the Syrian hamster are introduced. Recent in vitro cellular and molecular models have substantially increased the understanding of biliary tract calculi formation. Finally, a survey and new data about progesterone gene regulation of both cholesterol metabolism and gallstone formation obtained in the Syrian hamster model are compared with cholelithogenesis in human.

Ultrastructural aspects of human gallbladder epithelial cells in cholelithiasis: production of anionic mucus.

The surface epithelium of 28 gallbladders removed during elective cholecystectomies and pathology collection was studied ultrastructurally. Focusing on 10 of the 28 cases that were diagnosed as cholecystitis, we found that the epithelium displayed numerous apical mucous granules and bulging apical apices. Mucous granule changes included 1) hyperproduction of secretory granules of neutral type containing an electron-dense proteinaceous spherule, similar to that described in other mucus-producing glands of the digestive system, and 2) production of anionic, osmiophilic secretory mucus. Other alterations of the surface epithelial cells included the production of bizarre surface appendages resembling primitive cilia without axoneme and epithelial excrescences.

Flow cytometric and ultrastructural aspects of the synergistic antitumor activity of vitamin C-vitamin K3 combinations against human prostatic carcinoma cells.

Transmission and scanning electron microscopy and flow cytometry were employed to characterize the cytotoxic effects of vitamin C (VC), vitamin K3 (VK3), or VC-VK3 combinations on a human prostate carcinoma cell line (DU145) following a 1-h vitamin treatment and a 24-h incubation in culture medium. Cells exposed to VC exhibited membranous blebs, aberrant microvillar morphology, mitochondria with swollen cristae and intramitochondrial deposits, as well as nucleoli with segregated components. VK3-treated cells displayed a damaged cytoskeleton and membranes, a cytoplasm which contained large lumen, condensed polysomes, and severely damaged mitochondria with residual bodies, and nuclei which exhibited chromatic condensation, pyknosis, and karyolysis. VC-VK3-treated cells exhibited characteristics consistent with necrosis, i.e. swollen mitochondria and swollen, achromatic nuclei with marginated chromatin and intact envelopes, while other cells displayed characteristics consistent with apoptosis, i.e. expulsion of organelle-containing blebs, margination of nuclear chromatin, and segregation of nucleolar components. Vitamin treatment also decreased DNA synthesis, induced a S/G2 block in the cell cycle, and resulted in the accumulation of fragmented DNA. These results suggested that increased oxidative stress, subsequent membrane damage, and DNA fragmentation were responsible for enhanced cytotoxicity of the vitamin combination.