Irena Manov

Acetaminophen hepatotoxicity and mechanisms of its protection by N-acetylcysteine: a study of Hep3B cells

Acetaminophen (AAP) hepatotoxicity, resulting in centrilobular necrosis, is frequently encountered following suicidal attempts, especially by adolescents, but also after its excessive use in infants. The subcellular and molecular sequences leading to hepatocellular cell death are not yet clear. We therefore investigated AAP hepatotoxicity by using cultured hepatoma-derived cells (Hep3B) exposed to AAP and N-acetylcysteine (NAC), used as a protective agent. Specifically, we studied the role of apoptosis and oxidative damage as putative mechanisms of AAP-associated cytotoxicity. Hep3B cells were exposed to AAP (5-25 mM) and NAC (5 mM) for different time periods. Cell viability was assessed by the Alamar Blue Reduction Test and LDH. Oxidative damage was evaluated by measuring reactive oxygen species (ROS) and glutathione. AAP-induced apoptosis was investigated by flow cytometry and transmission electron microscopy. We found that: 1. In Hep3B cells, AAP causes a time- and concentration-dependent cytotoxic effect, leading to oxidative stress, mitochondrial dysfunction, alterations of membrane permeability and apoptosis; 2. In the course of AAP cytotoxicity, the generation of ROS appears as an early event which precedes decrease of viability, LDH leakage, glutathione depletion and apoptosis; 3. NAC protects Hep3B cells from AAP-induced oxidative injury, but does not prevent apoptosis.

Pronounced cancer resistance in a subterranean rodent, the blind mole-rat, Spalax: in vivo and in vitro evidence

BackgroundSubterranean blind mole rats (Spalax) are hypoxia tolerant (down to 3% O2), long lived (>20 years) rodents showing no clear signs of aging or aging related disorders. In 50 years of Spalax research, spontaneous tumors have never been recorded among thousands of individuals. Here we addressed the questions of (1) whether Spalax is resistant to chemically-induced tumorigenesis, and (2) whether normal fibroblasts isolated from Spalax possess tumor-suppressive activity.ResultsTreating animals with 3-Methylcholantrene (3MCA) and 7,12-Dimethylbenz(a) anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA), two potent carcinogens, confirmed Spalax high resistance to chemically induced cancers. While all mice and rats developed the expected tumors following treatment with both carcinogens, among Spalax no tumors were observed after DMBA/TPA treatment, while 3MCA induced benign fibroblastic proliferation in 2 Spalax individuals out of12, and only a single animal from the advanced age group developed malignancy 18 months post-treatment. The remaining animals are still healthy 30 months post-treatment. In vitro experiments showed an extraordinary ability of normal Spalax cultured fibroblasts to restrict malignant behavior in a broad spectrum of human-derived and in newly isolated Spalax 3MCA-induced cancer cell lines. Growth of cancer cells was inhibited by either direct interaction with Spalax fibroblasts or with soluble factors released into culture media and soft agar. This was accompanied by decreased cancer cell viability, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation and mitochondrial fragmentation. Cells from another cancer resistant subterranean mammal, the naked mole rat, were also tested for direct effect on cancer cells and, similar to Spalax, demonstrated anti-cancer activity. No effect on cancer cells was observed using fibroblasts from mouse, rat or Acomys. Spalax fibroblast conditioned media had no effect on proliferation of noncancerous cells.ConclusionsThis report provides pioneering evidence that Spalax is not only resistant to spontaneous cancer but also to experimentally induced cancer, and shows the unique ability of Spalax normal fibroblasts to inhibit growth and kill cancer cells, but not normal cells, either through direct fibroblast-cancer cell interaction or via soluble factors. Obviously, along with adaptation to hypoxia, Spalax has evolved efficient anti-cancer mechanisms yet to be elucidated. Exploring the molecular mechanisms allowing Spalax to survive in extreme environments and to escape cancer as well as to kill homologous and heterologous cancer cells may hold the key for understanding the molecular nature of host resistance to cancer and identify new anti-cancer strategies for treating humans.

Hepatotoxicity of anti-inflammatory and analgesic drugs: ultrastructural aspects

AbstractWith the increasing incidence of drug-induced liver disease, attempts are being made to better understand the mechanisms behind these frequently life-endangering reactions. Analgesics and anti-inflammatory drugs are a major group exhibiting hepatotoxicity. We review research relating to these reactions, focusing on ultrastructural findings, which may contribute to the comprehension and possible avoidance of drug-induced liver disease. We also present some original observations on clinical material and cultured cells exposed to acetaminophen alone or in combination with the antioxidant N-acetylcysteine or the P-glycoprotein inhibitor verapamil.

Inhibition of doxorubicin‐induced autophagy in hepatocellular carcinoma Hep3B cells by sorafenib – the role of extracellular signal‐regulated kinase counteraction

A multikinase inhibitor of the Raf/mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) pathway, sorafenib, is increasingly being used in the management of hepatocellular carcinoma, and its combination with conventional chemotherapeutics has stimulated particular interest. Although the combination of sorafenib with doxorubicin (DOX) is presently being investigated in a phase III randomized trial, little is known about the molecular mechanisms of their interaction. Because DOX causes cell death through upregulation of the MEK/ERK pathway, and sorafenib has an opposite influence on the same cascade, we hypothesized that co‐treatment with these drugs may lead to an antagonistic effect. DOX treatment arrested proliferation and induced autophagic cell death in Hep3B cells, whereas apoptotic changes were not conspicuous. Sorafenib alone affected viability and caused massive mitochondrial degradation. However, when added together with DOX, sorafenib facilitated cell cycle progression, increased survival, and reduced autophagy. To evaluate the molecular mechanisms of this phenomenon, we examined the expression of ERK1/2, protein kinase B (Akt), and cyclin D1, as well as the members of Bcl‐2 family. ERK1/2 activation induced by DOX was suppressed by sorafenib. Similarly, ERK targeting with the selective inhibitor U0126 impaired DOX‐induced toxicity. Treatment with sorafenib, either alone or in combination with DOX, resulted in Akt activation. The role of sorafenib‐induced degradation of cyclin D1 in the suppression of DOX efficiency is discussed. In conclusion, MEK/ERK counteraction, stimulation of survival via Akt and dysregulation of cyclin D1 could contribute to the escape from DOX‐induced autophagy and thus promote cancer cell survival. The use of MEK/ERK inhibitors in combination with chemotherapeutics, intended to enhance anticancer efficacy, requires the consideration of possible antagonistic effects.

Microvillous Inclusion Disease: Ultrastructural Variability

Microvillous inclusion disease (MVID) is a congenital, usually neonatal, autosomal recessive condition manifested by severe, prolonged secretory diarrhea. Intestinal biopsies reveal extensive microvilli abnormalities, typical inclusions and vesicles mainly of the apical-luminal enterocytes and colonocytes. Although diagnosis can be suspected by special stains of the mucosa (PAS, CD10), the definitive diagnosis, recommended in view of potential intestinal transplantation, requires electron microscopy. In view of the marked variability of ultrastructural changes, extensive illustration is considered valuable for diagnosis. While the pathogenesis is still unknown, a number of images illustrate the suspected “arrested-trafficking” hypothesis of microvillous abnormalities. Others micrographs support the “engulfing” mechanism of inclusion formation. The electron micrographs should help ultrastructural diagnosis in this heterogeneous disease and can confirm diagnosis even in the absence of the typical inclusions.

Infantile mitochondrial hepatopathy is a cardinal feature of MEGDEL syndrome (3-methylglutaconic aciduria type IV with sensorineural deafness, encephalopathy and Leigh-like syndrome) caused by novel mutations in SERAC1.

3‐Methylglutaconic aciduria (3‐MGCA) type IV is defined as a heterogeneous group of inborn errors featuring in common 3‐MGCA and associated with primary mitochondrial dysfunction leading to a spectrum of multisystem conditions. We studied four patients who presented at birth with a clinical picture simulating a primary mitochondrial hepatic disorder consistent with the MEGDEL syndrome including 3‐MGCA, sensorineural deafness, encephalopathy and a brain magnetic resonance imaging with signs of Leigh disease. All affected children displayed biochemical features consistent with mitochondrial OXPHOS dysfunction including hepatic mitochondrial DNA depletion in one patient. Homozygosity mapping identified a candidate locus on 6q25.2‐6q26. Using whole exome sequencing, we identified two novel homozygous mutations in SERAC1 recently reported to harbor mutations in MEGDEL syndrome. Both mutations were found to lead to decreased or absent expression of SERAC1. The present findings indicate that infantile hepatopathy is a cardinal feature of MEGDEL syndrome. We thus propose to rename the disease MEGDHEL syndrome.

The Liver in Congenital Disorders of Glycosylation: Ultrastructural Features

A new group of genetic diseases characterized by defective glycoprotein biosynthesis was recently described. Transferrin isoelectric focusing enabled identification of several types of patients with congenital disorders of glycosylation (CDG). The authors report on the liver involvement in two siblings with CDG type Ix presenting with failure to thrive and hypertransaminasemia who developed cardiomyopathy. In the initially affected infant, liver biopsy at 13 months of age showed increased periportal cellularity, steatosis, and mild fibrosis. Ultrastructurally, the hepatocytes displayed numerous myelinosomes, mostly with a pericanalicular polarization. No myelinosomes were seen in the bile canaliculi, Kupffer cells, and sinusoidal lining cells. Focal large droplet steatosis was also noticed. These ultrastructural findings represent another diagnostic element in this heterogenic group of conditions. Electron microscopy can contribute to the elucidation of hypertransaminasemia and differentiate some types of CDG from other lysosomal diseases.

Transcription Pattern of p53-Targeted DNA Repair Genes in the Hypoxia-Tolerant Subterranean Mole Rat Spalax

The tumor suppressor gene p53 induces growth arrest and/or apoptosis in response to DNA damage/hypoxia. Inactivation of p53 confers a selective advantage to tumor cells under a hypoxic microenvironment during tumor progression. The subterranean blind mole rat, Spalax, spends its life underground at low-oxygen tensions, hence developing a wide range of respiratory/molecular adaptations to hypoxic stress, including critical changes in p53 structure and signaling pathway. The highly conserved p53 Arg(R)-172 is substituted by lysine (K) in Spalax, identical with a tumor-associated mutation. Functionality assays revealed that Spalax p53 is unable to activate apoptotic target genes but is still capable of activating cell cycle arrest genes. Furthermore, we have shown that the transcription patterns of representative p53-induced genes (Apaf1 and Mdm2) in Spalax are influenced by hypoxia. Cell cycle arrest allows the cells to repair DNA damage via different DNA repair genes. We tested the transcription pattern of three p53-related DNA repair genes (p53R2, Mlh1, and Msh2) under normoxia and short-acute hypoxia in Spalax, C57BL/6 wild-type mice, and two strains of mutant C57BL/6 mice, each carrying a different mutation at the R172 position. Our results show that while wild-type/mutant mice exhibit strong hypoxia-induced reductions of repair gene transcript levels, no such inhibition is found in Spalax under hypoxia. Moreover, unlike mouse p53R2, Spalax p53R2 transcript levels are strongly elevated under hypoxia. These results suggest that critical repair functions, which are known to be inhibited under hypoxia in mice, remain active in Spalax, as part of its unique hypoxia tolerance mechanisms.

High-Dose Acetaminophen Inhibits the Lethal Effect of Doxorubicin in HepG2 Cells: The Role of P-glycoprotein and Mitogen-Activated Protein Kinase p44/42 Pathway

Doxorubicin (DOX) is a widely used chemotherapeutic drug for human hepatocellular carcinoma (HCC). A major limitation to its effectiveness is the development of multidrug resistance of cancer cells. In clinical trials, patients with advanced HCC were treated with high-dose acetaminophen (HAAP) in an effort to improve the antitumor activity of chemotherapeutics. In this study, we investigated the effect of concomitant treatment of DOX and HAAP on hepatoma-derived HepG2 cells. Viability, cell cycle distribution, and ultrastructure were examined. Unexpectedly, HAAP, when added to DOX-exposed cells, increased cell viability, released cell cycle arrest, and decreased apoptosis. To elucidate the mechanisms by which HAAP reduces the DOX lethal effect to HepG2 cells, we investigated the multidrug resistance P-glycoprotein (P-gp) and p44/42-mitogen-activated protein kinase (MAPK) pathways. The P-gp function was enhanced by DOX and HAAP, and it was further stimulated during combined treatment, leading to decreased DOX retention. Verapamil (VRP), when added to DOX + HAAP exposure, increased DOX accumulation and restored DOX-induced toxicity. The increased phospho-p44/42-MAPK level in DOX-exposed cells was inhibited by HAAP. In addition, suppression of p44/42 activation by the p44/42-MAPK inhibitor 2′-amino-3′-methoxyflavone (PD98059) blocked DOX-induced apoptosis. These findings suggest that the antagonistic effect of concomitant DOX + HAAP treatment occurs as a result of interactive stimulation of P-gp, generating decreased intracellular drug concentrations. Furthermore, inhibition of the p44/42-MAPK phosphorylation by HAAP could abolish the DOX-induced cell death pathway. Thus, combined treatment by DOX + HAAP, intended to improve chemotherapeutic efficacy, could have an opposite effect facilitating cancer cell survival.

Human Herpesvirus 8 in Primary Effusion Lymphoma in an HIV-Seronegative Male

BACKGROUND: AIDS-related body cavity-based lymphoma, or primary effusion lymphoma (PEL), is a distinct clinicopathologic entity that occurs predominantly in immunosuppressed patients infected with human herpesvirus 8 (HHV-8), also known as Kaposis sarcoma-associated herpesvirus. Although it rarely occurs in human immunodeficiency virus (HIV)-negative patients, we report such a case here. CASE: A 74-year-old male, who was HIV and Epstein-Barr virus (EBV) negative, was admitted to the hospital with dyspnea and chest pain. Chest radiography and computed tomography showed right pleural effusion. Cytologic analysis of the pleural effusion revealed a high grade lymphoma with round nuclei, prominent nucleoli and abundant cytoplasm. Polymerase chain reaction performed on the pleural effusion was positive for HHV-8 and negative for EBV. On molecular studies, the immunoglobulin heavy and kappa light chains were rearranged. Flow cytometry revealed a hyperploid fraction with DNA index of 1.29 expressing CD30. Immuno-staining for HHV-8 from a cell block was positive. Electron microscopy revealed lymphomalike cells, many in various stages of apoptosis, with large nucleoli and clusters of viruslike particles in the nucleoplasm. CONCLUSION: A firm diagnosis of PEL can be established by the examination ofcells from the lymphomatous effusion by a combination of cytology, molecular genetics, phenotypic features, immunostaining and electron microscopy. To our knowledge, this is the first case in which immunostaining for anti-HHV-8 monoclonal antibodies was used to support the diagnosis.