Martha E. Ramos-Márquez

Alpha-lipoic acid regulates heme oxygenase gene expression and nuclear Nrf2 activation as a mechanism of protection against arsenic exposure in HepG2 cells

Oxidative stress is a known mechanism induced, among other things, by arsenic toxicity. As a response, the cell triggers the synthesis of antioxidant and stress response elements like glutathione and heme oxygenase. Alpha-lipoic acid (ALA) is a well-known antioxidant that confers protection to oxidative stress conditions. We analyzed the effect of ALA pretreatment on Nrf2-responsive gene expression of HepG2 cells exposed to As(3+). Cells were treated with 5mM ALA and 8h later exposed to 50μM As(3+) for 24h, analyzing MTT-activity, glutathione content, Nrf2 induction and antioxidant gene expression. As(3+) increased glutathione (154%), heme oxygenase, glutamate cystein ligase, modifier subunit and metallothionein (35-fold, 10-fold and 9-fold, respectively). ALA prevented the strong expression of heme oxygenase by As(3+) exposure (from 35- to 5-times of control cells), which correlated with the reduction of Nrf2 observed in As(3+) group. ALA pretreatment can down-modulate the response mediated by Nrf2 and provide protection to As(3+) exposed HepG2 cells.

Truncated active matrix metalloproteinase-8 gene expression in HepG2 cells is active against native type I collagen

BACKGROUND/AIMS Excess type I collagen accumulation is a major feature of fibrotic diseases such as liver cirrhosis. Reversion of this disease has not been fully accomplished. Physiologically, collagen is degraded by interstitial collagenases, neutrophil collagenase (MMP-8) being the most active against type I collagen. Introduction of MMP-8 gene into liver cells could be an advantageous tool to potentiate fibrosis degradation. METHODS We cloned latent and active MMP-8 genes in prokaryotic and eukaryotic expression vectors and an adenoviral vector. Transfection of MMP-8 in HepG2 was effectuated by CaPO4, polylysine-lactose (P-L) and adenoviral transduction, and cells and culture supernatant were harvested 72 h after transfection for analysis of MMP-8 expression by reverse transcription-polymerase chain reaction and collagenolytic activity. RESULTS AND CONCLUSIONS We show that a truncated neutrophil collagenase (tMMP-8) lacking a portion of the carboxy terminus and with an intact aminoterminus (latent; l-tMMP-8) or a truncated amino terminus (active; a-tMMP-8) has enzymatic activity against native type I collagen, and the activity was inhibited by EDTA, 1,10-phenanthroline and TIMP-1. Both MMP-8 mRNA (latent and active) were detected by polymerase chain reaction in cells transfected with CaPO4, P-L and adenoviral transduction; however, relative expression of MMP-8 was enhanced when the plasmid was delivered as a P-L complex and increased by adenoviral infection. Finally, a-tMMP-8 cDNA was cloned in a vector under transcriptional control of a regulated promoter (PEPCK-a-tMMP-8). HepG2 cells transfected with the PEPCK-a-tMMP-8 plasmid DNA up-regulated expression of a-tMMP-8 after incubation of the cells with butyryl-cAMP and glucagon, while stimulation with insulin slightly down-regulated its expression. Recombinant MMP-8 expressed by HepG2-transduced cells can efficiently degrade soluble type I collagen, which is potentially useful for gene transfer therapies.

Unusual branchial arch, dermoepidermal and nervous system anomalies in a neonate with VACTERL-H syndrome

RESUMEN El sindrome VACTERL-H es un trastorno complejo de malformaciones congenitas que implica vertebras, ano, corazon, traquea, esofago, rinones, extremidades (del ingles limbs) e hidrocefalia. Su etiologia se ha identificado solo en algunos pacientes debido, en gran medida, a su naturaleza esporadica, asi como a su alto grado de heterogeneidad clinica. En este informe se presenta a un neonato con el sindrome VACTERL-H, al que se asocian anomalias inusuales del arco branquial, dermoepidermicas y del sistema nervioso, que se comparan con las descritas en la bibliografia medica. Segun nuestra experiencia, esta presentacion no solo amplia el conocimiento del espectro de anomalias que se puede presentar en el sindrome VACTERL-H, sino que tambien podria ser util en la identificacion de pacientes con este fenotipo heterogeneo. Palabras clave: sindrome VACTERL-H, heterogeneo, anomalias arco branquial. SUMMARY VACTERL-H syndrome is a complex disorder of congenital malformations that implies vertebrae, anus, heart, trachea, esophagus, kidneys, limbs and hydrocephalus. Its etiology has been identified in a fraction of patients largely due to their sporadic nature and its high degree of clinical heterogeneity. This report presents a newborn with VACTERL-H syndrome, associated with unusual branchial arch, dermoepidermal and nervous system anomalies, which are compared with those described in the medical literature. Based on our experience, the presentation of this case not only expands the knowledge of the spectrum of anomalies that can occur in VACTERL-H syndrome, but also can be useful in identifying patients with this heterogeneous phenotype.

Asociación VACTERL-H con agenesia tibial. ¿Expansión del fenotipo clínico?

4. Eisinger J, Flores J, Tyson JA, Shohet SB. Fluorescent cytoplasm and Hein bodies of haemoglobin Könl erythrocytes: evidence for intracellular heme catabolism. Blood. 1985;65:886. 5. Katoh R, Miyake T, Arai T. Unexpectedly low pulse oximeter readings in a boy with unstable haemoglobin Köln. Anesthesiology. 1994;80:472--4. 6. Gottschalk A, Silverberg M. An unexpected finding with pulseoximetry in a patient with haemoglobin Köln. Anesthesiology. 1994;80:472--4.

Current antioxidant molecular therapies for oxidative stress-related ailments.

Oxidative cell damage is a natural occurring phenomenon due to the aerobic conditions where cells are embedded; however, such injury can efficiently be controlled and repaired by the inherent antioxidants of the cell. When the oxidant/antioxidant balance is disrupted towards the former by any chemical, biological or physical insult a pathological state could be developed, therefore, an extensive list of compounds with proved or assumed antioxidants properties has largely been used for improving or restoring the health status. Pharmacological therapies as well as dietary or complementary therapies are continuously being investigated for the counteracting of the harmful or damaging effects of oxidation in cells or tissues. However, critical antioxidant levels are not always achieved at the target damaged cells by these approaches; on the other side, the expression of recombinant antioxidant genes specifically directed to the afflicted cell by gene delivery has shown remarkable results. In this review we summarize the literature focused on some of the current antioxidant molecular pharmacological strategies with particular emphasis to the gene transfer protocols involved in the treatment of oxidative stress-related disorders.

Ductular hyperplasia is characterized by an over expression of c-Myc in bile duct ligation + furan injured rats: possible role of interleukin-6

The aim of this study was focused on the analysis of gene expression of pro- and anti-inflammatory cytokines, and c-Myc in liver carcinogenesis in bile duct ligation (BDL)+furan treated rats. We also correlated the molecular and immunohistochemical findings with the degree of architecture distortion and histopathological changes in the liver. Groups of rats were subjected to BDL and one week later, animals were given furan in corn oil by gavage at 45 mg/kg body weight, once a week, five times weekly, for 1--4 weeks. Determination of pro- and anti-inflammatory cytokines gene expression by reverse transcriptase-polymerase chain reaction (RT-PCR) was performed in tissue, and liver sections were processed for immunohistochemistry of c-Myc. There was a significant increase in the TNF-alpha gene expression at the first week after BDL+furan treatment. Interleukin-6 gene expression was also increased at the first week and remained elevated up to the third week after treatment. C-Myc expression was detected beginning at the first week and peaking at fourth week after treatment where its expression was found at histologically distorted parenchymal areas. The present study suggest a possible relationship in the overexpression of pro-inflammatory cytokines and c-Myc in the development of carcinogenesis.

Association of Paraoxonase-1 Q192R (rs662) Single Nucleotide Variation with Cardiovascular Risk in Coffee Harvesters of Central Colombia

Paraoxonase 1 (PON1), a high-density lipoprotein-associated antioxidant enzyme, hydrolyzes several organophosphate pesticides and oxidized lipids. The PON1 Q192R polymorphism affects the catalytic efficiency and is considered a risk factor for pesticide intoxication and cardiovascular disease (CVD) but the association is not consistent between individuals or populations. We aimed to study the association of PON1 Q192R polymorphism with CVD risk in coffee harvesters of central Colombia. Demographics were collected from 205 subjects via standardized questionnaires. Lipid profiles and serum butyrylcholinesterase (BChE) were measured by standard procedures. The calculated 10-year atherosclerotic CVD (ASCVD) risk was used as the cardiovascular risk estimate. Q192R genotype was determined by real-time PCR. Prevalence of hypertension, hypercholesterolemia, and the 10-year ASCVD risk was 33%, 62%, and 22%, respectively. BChE levels were no indicative of recent pesticide exposure, although a positive correlation was observed with BChE and hypercholesterolemia. The Q192R genotype frequencies were 38% (QQ), 44% (QR), and 18% (RR). We found an association of the 192Q genotype with hypertension. The results of this study signal the importance to evaluate the influence and potential interactions of BChE and PON1 192Q allele with known genetic and environmental factors implicated in the pathogenesis of CVD.