Genotoxic effects of ethylene oxide,propylene oxide and epichlorohydrin in humans: update review (1990-2001)

Ethylene oxide (EtO), propylene oxide (PO) and epichlorohydrin (ECH) are important industrial chemicals widely used as intermediates for various synthetic products. EtO and PO are also environmental pollutants. In this review we summarize data published during the period 1990-2001 concerning both the genotoxic and carcinogenic effects of these epoxides in humans. The use of DNA and hemoglobin adducts as biomarkers of exposure and the role of polymorphism, as well as confounding factors, are discussed. We have also included recent in vitro data comprising genotoxic effects induced by EtO, PO and ECH in mammalian cells. The uncertainties regarding cancer risk estimation still persist, in spite of the large database collected.     

Increased anthocyanin accumulation in aster flowers at elevated temperatures due to magnesium treatment

Temperature is one of the main external factors affecting anthocyanin accumulation in plant tissues: low temperatures cause an increase and elevated temperatures cause a decrease in anthocyanin concentration. Several metals have been shown to increase the half-life time of anthocyanins, by forming complexes with them. We studied the combined effect of elevated temperatures and increased metal concentrations on the accumulation of anthocyanins in aster 'Sungal' flowers. It has been found that magnesium treatment of aster plants or detached flower buds, partially prevents colour fading at elevated temperatures. Anthocyanin concentration of aster 'Sungal' flowers grown at 29°C/21°C day/night, respectively, was about half that of flowers grown at 17°C/9°C. The activity of phenylalanine ammonia-lyase (PAL) and chalcone isomerase (CHI) decreased as the temperature increased. Treatment of both whole plants and detached flower buds grown at elevated temperatures in the presence of magnesium salts, increased flower anthocyanin concentration by up to 80%. Measurement of magnesium following these treatments revealed an increased level of the metal in the petals, suggesting a direct effect. Magnesium treatment does not seem to cause increased synthesis of anthocyanin through a stress-related reaction, since the activities of both PAL and CHI did not increase due to this treatment. The results of this study show that increasing magnesium levels in aster petals prevents the deleterious effect of elevated temperatures on anthocyanin accumulation, thus enhancing flower colour.     

Apoptosis during ectromelia orthopoxvirus infection is DEVDase dependent: in vitro and in vivo studies

Ectromelia virus (EV), which causes mousepox, is a member of the orthopoxviruses that are defined as being able to suppress apoptosis. Caspase-3 is one of the key effector proteases which regulates the apoptotic cascade and which is responsible for DNA fragmentation observed during apoptosis. It is well known that viruses, especially poxviruses, can inhibit caspase activity. Here, we report that EV can regulate apoptosis in vitro, suppressing the activity of caspases recognizing the DEVD (Asp-Glu-Val-Asp) motif (caspase-3 and -7) before successful virus replication is completed. Caspase-3 activity measurement showed that an increase in caspase-3 activity preceded the peak of DNA fragmentation demonstrated by TUNEL staining of L929 and RK-13 cells. By using specific caspase inhibitors (Ac-DEVD-CHO, Ac-IETD-CHO and zVAD-fmk), we showed that caspase-3 and -7 (DEVDases) are major effector caspases during EV-induced apoptosis in permissive L929 and RK-13 cell cultures. Apoptosis in vivo seems to play an important role during viraemia as well as during the clearance of EV from genetically susceptible BALB/c (H-2(d)) mice. However, as shown by measurement of caspase-3 activity, caspase-3 protein detection and M30-antibody staining, both DEVDases seem to play an important role during EV clearance from draining lymph nodes and conjunctivae at 15 days p.i. up to 20 days p.i., whereas in the liver and spleen DNA fragmentation coexisted with viral multiplication and secondary viraemia. Apoptosis was DEVDase dependent only in the liver, while spleen DNA fragmentation observed between 5 and 10 days p.i. was caspase independent. Therefore, we conclude that DEVDase- (caspase-3- and caspase-7-) dependent apoptosis is an important mechanism regulating the resolution of EV infection.     

Non-radioactive detection of β-glucuronidase and chloramphenicol acetyltransferase activities in co-transformed protoplasts by HPLC

Summary The use of transient gene expression assays for the study of natural or engineered plant promoters is affected by a considerable degree of inter-experiment variability. As a means of obtaining interpretable data from a limited number of experiments, we worked out conditions for the simultaneous determi nation of the activity of two reporter genes, a sample and a reference, ona single extract of co-transformed protoplasts. ß-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) genes, both under the control of the CaMV 35S promoter, were transferred into tobacco (Nicotiana tabacum L.) protoplasts on two independent plasmids. The parallel expression of the two reporter genes in several independent co-transformation experiments was verified. Conditions for the use of a single protoplast extraction buffer and for the simultaneous assay of both reporter gene activities were set up. A HPLC method for the non-radioactive determination of both enzyme activities on a single aliquot of the reaction mixture was developed. The resulting procedure was tested using the GUS gene as reference and the CAT gene, under the control of either wild type or upstream-deleted (–90) CaMV 35S promoter, as sample. The protocol is simple and allows the fast analysis of plant promoters in the presence of a true internal standard under conditions in which assay manipulations are reduced to a minimum and both reporter gene activities are subjected to the same experimental treatments.Abbreviations CaMV cauliflower mosaic virus - CAT chloramphenicol acetyl transferase - EDTA ethylenediaminetetraacetic acid - GUS ß-glucuronidase - HPLC high performance liquid chromatography - MES 2-morpholinoethanesulphonic acid - MS medium after Murashige and Skoog (1962) - MUG 4-methyl umbelliferyl glucuronide - MU methylumbelliferone - NOS nopaline synthase - PEG polyethylene glycol - TRIS tris-hydroxymethyl aminomethane - UV ultraviolet     

Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

The presence of 1.0mm-dibutyryl cyclic AMP (N(6),O(2')-dibutyryladenosine 3':5'-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24-36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [(3)H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [(3)H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [(14)C]fucose and [(3)H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse-chased with [(14)C]fucose and [(3)H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins.     

Structural analysis of denaturant-protein interactions: Comparison between the effects of bromoethanol and SDS on denaturation and renaturation of triclinic lysozyme

This paper summarizes our crystallographic studies of the interaction of denaturants with cross-linked triclinic lysozyme. Electron density maps of various bromoethanol-lysozyme complexes are analyzed and compared to those reported earlier for SDS-lysozyme complexes. Despite differences in the chemical nature and size of the two denaturants their mode of interaction with the protein is quite similar, suggesting the existence of a general mechanism for binding of hydrophobic-hydrophilic denaturants to proteins. Our results are consistent with the conclusion that lysozyme consists of two domains connected by a flexible segment and that this segment represents an internal degree of freedom of the protein.The work was carried out during the tenure of a fellowship from the European Molecular Biology OrganizationWe are grateful to Dr. Gerson Cohen for providing us with his data processing programs, to Drs. David Haas, Paul Sigler, Thomas Creighton and Micael James for helpful discussions, and to Mr. Samuel Getteno for his invaluable technical assistance.     

III. Nitric oxide mediates the action of lipoteichoic acid on the function of human urothelial cells

Gram-positive bacteria are recognized pathogens in urinary tract infections. Lipoteichoic acids, major components of the cell wall of gram-positive bacteria, are important virulence attributes, but their mechanism of action is not well understood. We have postulated that infection-induced altered function of progenitors of urothelial cells (UT) residing in the basal layer is likely to have long-lasting effects on the architecture and function of the urothelium. Our earlier in vitro studies in UT of basal type, grown under growth restricting conditions, have shown that (1) treatment with lipoteichoic acid from Streptococcus faecalis (LT-2) stimulates a subpopulation of progenitors of urothelial cells to proliferate, and (2) resulting large colonies differentiated at an increased rate under conditions simulating those in the basal layer of the urothelium. The hypothesis underlying the present studies was that nitric oxide (NO) mediated LT-2 action on these functions of UT. Immunocytochemical studies using an antibody against inducible nitric oxide synthase (iNOS) confirmed expression of iNOS in LT-2-treated UT. Our hypothesis was tested by treating UT grown under growth restricting conditions (0.005% bovine pituitary extract) with LT-2 (25 μg/ml), in the presence or absence of inhibitors of NOS (1 mM N^G-nitro-L-arginine methyl ester [L-NAME]; 1 μM dexamethasone [DEXA]) or 25 μM hemoglobin, a potent inactivator of NO. Treatment with LT-2 in the presence of these agents prevented the following effects of LT-2 alone: (1) the stimulatory effect on proliferation of single cells, as well as within the resulting large colonies; (2) the subsequent differentiation of large colonies resulting from this proliferative activity, as indicated by distribution of β₁ subunit-containing integrins to cell-cell contacts; (3) the inhibitory effect on the subsequent ability of LT-2-treated UT to attach to extracellular matrix proteins. These studies suggest that induction of NOS by LT-2, initially aimed at restricting the replication of infectious agents, may have potential cost of damage to the host bladder by interfering with urothelial differentiation. © 1996 Wiley-Liss, Inc.