Pharmacological inhibition of guanosine triphosphate cyclohydrolase1 elevates tyrosine phosphorylation of caveolin1 and cellular senescence.

The role of 2,4-diamino-6-hydroxypyrimidine (DAHP), on cellular-senescence remains unclear as differential effects of DAHP have been reported in cardiovascular and cerebrovascular systems. We investigated the effect of pharmacologically-induced guanosine-triphosphate-cyclohydrolase1 (GTPCH1)-inhibition, through DAHP, on cellular-senescence in experimentally-induced diabetic and non-diabetic Wistar rats. Cellular-senescence was evaluated through senescence-associated events, namely, cell-cycle-arrest of peripheral blood mononuclear cells (PBMNCs); myocardial DNA fragmentation, total antioxidant capacity (TAC), telomerase-activity, nicotinamide adenine dinucleotide (NAD+)-content and tyrosine14-phosphorylation of caveolin1 (pY14) in similarly-aged, pubertal Wistar rats with streptozotocin (STZ) and/or DAHP. Oxidative stress (OS) indices such as myocardial biopterin concentrations (tetrahydrobiopterin-BH4 and dihydrobiopterin-BH2) and plasma total nitrite and nitrate (NOx) were determined. DAHP, per se, exhibited distinct senescence; in addition, in STZ+DAHP (the cardiomyopathy model), there was a marked accumulation of cells in G0G1 phase, as evidenced through flow-cytometry analysis, as-well-as fragmented DNA, than the respective controls suggesting the DAHP-mediated onset of senescence in circulating cells and the myocardium, with or without STZ. Concentrations of BH4 and BH2, and NOx were impaired in STZ and/or DAHP, indicating elevated OS in the treatment groups. In the independent treatment groups or the combination treatment, typical senescence indicators including myocardial telomerase-activity, NAD+-content and TAC were significantly reduced, while there was a marked elevation in the concentrations of pY14 as compared to the respective controls, reinforcing the occurrence of senescence in PBMNCs and the myocardium. We postulate that DAHP promotes early onset of cellular-senescence, potentially through OS-mediated cellular events in diabetic or non-diabetic models.

A Guardian Angel Phosphatase for Mainline Carbon Metabolism.

It is increasingly clear that many metabolic enzymes mistakenly form minor but toxic side-products that must be eliminated to maintain normal fluxes. Collard et al. show that this is true of two iconic glycolytic enzymes, and that a hitherto somewhat mysterious phosphatase rescues central carbon metabolism from their mistakes.

A conserved phosphatase destroys toxic glycolytic side products in mammals and yeast.

Metabolic enzymes are very specific. However, most of them show weak side activities toward compounds that are structurally related to their physiological substrates, thereby producing side products that may be toxic. In some cases, 'metabolite repair enzymes' eliminating side products have been identified. We show that mammalian glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase, two core glycolytic enzymes, produce 4-phosphoerythronate and 2-phospho-L-lactate, respectively. 4-Phosphoerythronate strongly inhibits an enzyme of the pentose phosphate pathway, whereas 2-phospho-L-lactate inhibits the enzyme producing the glycolytic activator fructose 2,6-bisphosphate. We discovered that a single, widely conserved enzyme, known as phosphoglycolate phosphatase (PGP) in mammals, dephosphorylates both 4-phosphoerythronate and 2-phospho-L-lactate, thereby preventing a block in the pentose phosphate pathway and glycolysis. Its yeast ortholog, Pho13, similarly dephosphorylates 4-phosphoerythronate and 2-phosphoglycolate, a side product of pyruvate kinase. Our work illustrates how metabolite repair enzymes can make up for the limited specificity of metabolic enzymes and permit high flux in central metabolic pathways.

The mutagenicity of thymidine glycol in Escherichia coli is increased when it is part of a tandem lesion.

Tandem lesions are comprised of two contiguously damaged nucleotides. Tandem lesions make up the major family of reaction products generated from a pyrimidine nucleobase radical, which are formed in large amounts by ionizing radiation. One of these tandem lesions contains a thymidine glycol lesion flanked on its 5'-side by 2-deoxyribonolactone (LTg). The replication of this tandem lesion was investigated in Escherichia coli using single-stranded genomes. LTg is a much more potent replication block than thymidine glycol and is bypassed only under SOS-induced conditions. The adjacent thymidine glycol does not significantly affect nucleotide incorporation opposite 2-deoxyribonolactone in wild-type cells. In contrast, the misinsertion frequency opposite thymidine glycol, which is negligible in the absence of 2-deoxyribonolactone, increases to 10% in wild-type cells when LTg is flanked by a 3'-dG. Experiments in which the flanking nucleotides are varied and in cells lacking one of the SOS-induced bypass polymerases indicate that the mutations are due to a mechanism in which the primer misaligns prior to bypassing the lesion, which allows for an additional nucleotide to be incorporated across from the 3'-flanking nucleotide. Subsequent realignment and extension results in the observed mutations. DNA polymerases II and IV are responsible for misalignment induced mutations and compete with DNA polymerase V which reads through the tandem lesion. These experiments reveal that incorporation of the thymidine glycol into a tandem lesion indirectly induces increases in mutations by blocking replication, which enables the misalignment-realignment mechanism to compete with direct bypass by DNA polymerase V.

An improved bioassay for the detection of Bacillus thuringiensis beta-exotoxin.

Some Bacillus thuringiensis strains secrete beta-exotoxin, which is an insecticidal, thermostable adenine nucleotide analogue. Discrepancies between detection of beta-exotoxin by high-performance liquid chromatography and insect bioassays have shown the importance of bioassays in the determination of beta-exotoxin production. With the aim of improving the fly beta-exotoxin bioassay, a range of fly diets were evaluated and the best performing diet was incorporated into a novel beta-exotoxin bioassay. The improved bioassay is characterised by good control pupation percentages, low variability, easy setup and monitoring. The bioassay allowed unambiguous differentiation between beta-exotoxin producing and non-producing strains, and is suitable for the routine screening of B. thuringiensis strains for beta-exotoxins.

Role of oxidative stress in thuringiensin-induced pulmonary toxicity.

To understand the effect of thuringiensin on the lungs tissues, male Sprague-Dawley rats were administrated with thuringiensin by intratracheal instillation at doses 0.8, 1.6 and 3.2 mg/kg of body weight, respectively. The rats were sacrificed 4 h after treatment, and lungs were isolated and examined. Subsequently, an effective dose of 1.6 mg/kg was selected for the time course study (4, 8, 12, and 24 h). Intratracheal instillation of thuringiensin resulted in lung damage, as evidenced by increase in lung weight and decrease in alkaline phosphatase (10-54%), an enzyme localized primarily in pulmonary alveolar type II epithelial cells. Furthermore, the administration of thuringiensin caused increases in lipid peroxidation (21-105%), the indices of lung injury. In addition, the superoxide dismutase (SOD) and glutathione (GSH) activities of lung tissue extracts were measured to evaluate the effect of thuringiensin on antioxidant defense system. The SOD activity and GSH content in lung showed significant decreases in a dose-related manner with 11-21% and 15-37%, respectively. Those were further supported by the release of proinflammatory cytokines, as indicated by increases in IL-1beta (229-1017%) and TNF-alpha (234%) levels. Therefore, the results demonstrated that changes in the pulmonary oxidative-antioxidative status might play an important role in the thuringiensin-induced lung injury.

Pulmonary toxicity of thuringiensin administered intratracheally in Sprague-Dawley rats.

The purpose of this work is to evaluate the pulmonary toxicity of purified thuringiensin in Sprague-Dawley rats. Rats were intratracheally instillated with 0, 0.4, 0.8, 1.6, 3.2, 6.4 and 9.6 mg/kg body weight of thuringiensin. The results indicated that the acute pulmonary LD(50) of thuringiensin for rats was 4.4 mg/kg. The total number of inflammatory cells and lactate dehydrogenase (LDH) activity in the bronchoalveolar lavage (BAL) fluid increased in a dose-dependent manner after thuringiensin instillation. Furthermore, an effective dose of 1.6 mg/kg was selected for the time course study of pulmonary toxicity. The treated animals showed a significant increase in the weights of the lungs, hydroxyproline levels in the lungs and total number of cells in BAL fluid 2, 4, 7, 14, 28 and 56 days after treatment. In comparison with the control, the total protein concentrations in BAL fluid were increased by 361, 615, 116, 41, 34 and 41%, after 2, 4, 7, 14, 28 and 56 days, respectively. The LDH activity in BAL fluid showed a significant increase after 1, 2, 4, 7, 14, 28 and 56 days. The increases in fibronectin levels were 164, 552, 490, 769, 335, 257 and 61% at the corresponding times, but neither tumor necrosis factor nor interleukin-1 increased. The treated rats presented abnormal histology including distributed inflammation in the bronchioles and alveoli, bronchial cellular necrosis on days 1 and 2, and areas of septal thickening with cellular infiltration and collagen deposit in the intestinal and alveolar spaces on days 4-56. Based on these biochemical and pathological parameters, intratracheal instillation of purified thuringiensin might cause significant pulmonary toxicity in rats.

Update on the detection of beta-exotoxin in Bacillus thuringiensis strains by HPLC analysis.

AIMS: The current work aimed to study the presence of beta-exotoxin by high-performance liquid chromatography (HPLC) in supernatant fluids from final whole cultures of the 69 type strains and 13 subtypes of Bacillus thuringiensis strains, as well as from some insecticidal strains. METHODS AND RESULTS: Results from HPLC and bioassays with Ephestia kuhniella (Lepidoptera Pyralidae) were compared. Type I beta-exotoxin was only detected in type strains representing serotypes H1, H9 and H10a,10b. Discrepancies between HPLC and bioassays were found in H8a,8b and some insecticidal strains, which suggests the occurrence of another soluble toxin different from type I beta-exotoxin, possibly type II beta-exotoxin. CONCLUSION: This study shows the need to use bioassays to determine the presence of beta-exotoxin activity. However, HPLC is a fast and sensitive technique if only type I beta-exotoxin is to be determined. The occurrence of beta-exotoxin in a type strain does not imply production of this metabolite by other strains belonging to the same serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: These results complete the characterization of type strains belonging to the International Entomopathogenic Bacillus Collection (Institut Pasteur, Paris, France).

Cellular inactivation induced by a radiopharmaceutical kit: role of stannous chloride.

Stannous chloride (SnCl2) has been used in many sectors of human activities such as food manufacturing and in nuclear medicine to produce radiopharmaceuticals labeled with technetium-99m (99mTc). Due to its importance and genotoxic potentiality, we decided to evaluate the biological effect induced by a nuclear medicine kit, which includes SnCl2, in association with glucoheptonic acid (GHA) which is employed for brain and renal scintigraphies. These studies were carried out with the Escherichia coli AB1157 strain and the deoxyribonucleic acid (DNA) plasmid pUC 9.1. The experiments, with different concentrations of SnCl2 and GHA, show an inverse relationship between both agents. When the GHA concentration was increased, the cellular inactivation induced by SnCl2 was reduced, as measured by the number of viable cells. Moreover, GHA protects the DNA molecule against the damage induced by SnCl2.

Stannous chloride and the glucoheptonic acid effect: study of a kit used in nuclear medicine.

Stannous dichloride is used as a reducing agent in the preparation of technetium-99m radiopharmaceuticals. We decided to evaluate the genotoxic potential of the tin (II)-glucoheptonate complex in the kit using a tester strain of Escherichia coli AB1157. Our results show that tin (II) chloride and the tin (II)-glucoheptonate complex exert a genotoxic effect in this system. While the genotoxic effect disappeared when the glucoheptonate concentration was increased, the glucoheptonate did not protect the cultures from the damaging effects of hydrogen peroxide. The ability of glucoheptonate to protect cultures from tin (II)-induced damage can be explained on the basis of its metal chelating properties.

Safety testing of Bacillus thuringiensis preparations, including thuringiensin, using the Salmonella assay.

The aim of this study was to test genotoxicity aspects of the safety of two strains of Bacillus thuringiensis. The strains were of serotype H-1, producing thuringiensin, toxic to flies, and serotype H-14, producing endotoxin, toxic to mosquitoes, but not thuringiensin. Four preparations were tested: Tenfold concentrated cell-free culture media of serotypes H-1 and H-14, prepurified thuringiensin, and purified endotoxin. No increases in revertant colony numbers of the tester strains of Salmonella typhimurium TA98 or TA100 were observed at the dose levels used with or without metabolic activation. Infrequent slight increases in revertant colony numbers in strains TA98 and TA1538 at a very high test dose of 50 mg/plate, both in the presence and absence of thuringiensin, were probably caused by histidine present in the growth medium of B. thuringiensis. Furthermore, the effects were at most slightly dose related and not reproducible, and therefore the preparations can be considered nonmutagenic.

Effect of Bacillus thuringiensis beta exotoxin on ultrastructures of midgut cells of Culex sitiens.

In midgut cells of Culex sitiens intoxicated with beta exotoxin of Bacillus thuringiensis, the microvilli are greatly reduced. In the cytoplasm there is progressive disarrangement of the membranes of the rough endoplasmic reticulum, first to spherules, and later to a granular mass. Minute vacuoles appearing in the cytoplasm are parts of the distorted labyrinth and remains of the Golgi membranes. There is no disintegration of the outer cell membrane, and no hypertrophy of goblet cells. Nuclei remain in their posterior position and there are no cytoplasmic autophagic vacuoles in the centre. Cells in the same part of the midgut differ in the degree of disorganization.

Increase in the resistance to the Bacillus thuringiensis supernatant effect in a Drosophila melanogaster wild type Oregon R line.

We report here the genetical and X chromosome rDNA molecular study of two Drosophila melanogaster Oregon R lines. These lines differ extensively in the degree of resistance of the females both to the lethal effect of an increased temperature and to that of Bacillus thuringiensis beta-exotoxin, which is an inhibitor of the nucleolar RNA polymerase. The 3B line, whose females are resistant, came from an Oregon R population subjected over several generations to increased temperature, 28 degrees C or over, while the other line is derived from the initial stock. Twofold variation was observed in the total number of ribosomal genes between the two lines. This variation applied to most ribosomal units, including the active ones. Variations among X chromosome rDNA content in a wild type population have thus been revealed using tests of resistance to the Bacillus thuringiensis beta-exotoxin. Additive variations in specific unit types between the two lines indicate that modifications to the rDNA content are not rare events.

Enhancement by phenothiazine and 2,5-di-O-acetyl-D-glucosaccharo-(1,4)(6,3)-dilactone of bladder carcinogenicity of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide in rats.

Rats concomitantly fed N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) and phenothiazine, or concomitantly fed FANFT and Glucaron then fed Glucaron alone had significantly greater incidences of transitional cell carcinomas of the bladder than rats fed FANFT alone. Caffeine and cysteamine did not affect FANFT bladder carcinogenesis. Phenothiazine induced nitroreductase activity of hepatic microsomes.

A subacute toxicity study on 99m Tc stannous Glucoheptonate injection.

A subacute toxicity study on 99m Tc stannous glucoheptonate was performed with rats, dogs and rabbits, injected intravenously at ten to 100 times the human dose on a body weight basis. There were no abnormalities in the clinical status of any of the animals. No changes were found in urinalysis, blood chemistry or hematology in the rabbit nor in gross examination, renal histology or bone marrow smears in the rats and rabbits. Hepatic histology was also done. A focal area of necrosis in a liver of one rabbit that had been injected with 100 times the human dose was observed using light microscopy. Examination by electron microscopy in another group of rats and rabbits was prompted by the observation of that lesion. This revealed vacuolated and dilated smooth endoplasmic reticulum and degranulated and vesiculated rough endoplasmic reticulum in all the test livers. X-ray microanalysis indicates that the ultrastructural changes are linked to the deposition of tin.