The contrast agent 2,3,5-triiodobenzoic acid (TIBA) induces cell death in tumor cells through the generation of reactive oxygen species.

The 2,3,5-triiodobenzoic acid (TIBA) is an iodine contrast agent used for visualization of tissue in X-ray techniques. However, TIBA induces physiological complications like increase in oxygen reactive species (ROS), and consequently, contrast-induced nephropathies. TIBA's antitumor activity was demonstrated in lung cancer, but the subcellular mechanisms involving its activity in tumor cells are still unknown. Thus, the objective of this work was evaluate whether the anti-tumor activity of TIBA involves ROS increase, in tumor lines of non-small cell lung cancer (H460), chronic myeloid leukemia (K562), and its cytotoxicity in normal renal epithelial (VERO). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay was used for evaluation of cell viability, the H2DCFDA (cell-permeant 2',7'-dichlorodihydrofluorescein diacetate) fluorescent probe to evaluate ROS induction, cell cycle analysis was performed using flow cytometry to measure cell death, and immunofluorescence with annexin/7-AAD (7-amino-actinomycin D), to assess the association of cell death with the ROS generation. TIBA decreases cell viability in a dose-dependent manner for the H460 and K562. However, VERO cells showed less response to the drug, with 70% viable cells after 72 h of treatment in the highest concentration of the drug. While the tumor cells with only 20% viable cells. Besides, tumor cells exhibited higher DNA fragmentation, compared to the renal line (VERO with 5% of fragmented DNA, H460 with 26%, and 56% in K562). Finally, TIBA-induced ROS increase and apoptosis in all lines, which is significantly decreased after treatment with the antioxidant N-acetyl-cysteine (NAC). These data demonstrate the relationship between the increased cellular oxidative stress and the anti-tumor action of the TIBA.

Biodistribution and Toxicity of X-Ray Iodinated Contrast Agent in Nano-emulsions in Function of Their Size.

PURPOSE: This study aimed to investigate the impact of the size of X-ray iodinated contrast agent in nano-emulsions, on their toxicity and fate in vivo. METHODS: A new compound, triiodobenzoate cholecalciferol, was synthetized, formulated as nano-emulsions, and followed after i.v. administration in mice by X-ray imaging (micro computed tomography). Physicochemical characterization and process optimization allowed identifying a good compromise between X-ray contrasting properties, monodispersity and stability. This also allowed selecting two formulations with different sizes, hydrodynamic diameters of 55 and 100 nm, but exactly the same composition. In vitro experiments were performed on two cell lines, namely hepatocytes (BNL-CL2) and macrophages (RAW264.7). RESULTS: Cell viability studies, cell uptake observations by confocal microscopy, and uptake quantification by fluorimetry, disclosed clear differences between two formulations, as well as between two types of cell lines. After i.v. injection of the two iodinated nano-emulsions in mice, CT scans provided the quantification of the pharmacokinetics and biodistributions. We finally showed that the size in the nano-emulsions has not a real impact on the pharmacokinetics and biodistributions, but has a strong influence on their toxicity, corroborating the in vitro results. CONCLUSIONS: This study shows that the size of the nanocarrier significantly matters, likely due to highly different interactions with cells and tissues. Graphical Abstract A study on the effect of the size of cholecciferol nano-emulsions, on their in vivo becoming, through X-ray imaging modality.

Indole-3-acetic acid reverses the harpin-induced hypersensitive response and alters the expression of hypersensitive-response-related genes in tobacco.

Harpin proteins stimulate hypersensitive response (HR) in plants. However, the mechanism by which HR is regulated is not clear. The role of the auxin, indole-3-acetic acid (IAA), in the control of harpin-stimulated HR was investigated. IAA was used to inhibit HR that was stimulated by purified fusion harpin(Xoo) protein in tobacco. Semi-quantitative PCR and qRT-PCR were employed to detect the expression of HR related genes. IAA at 100 µM reversed harpin-induced HR which was inhibited by 500 µM 2,3,5-triiodobenzoic acid (TIBA). Semi-quantitative PCR and qRT-PCR showed the combined application of 100 µM IAA and harpin protein from Xanthomonas oryzae enhanced the expression of HR marker gene, hsr203J, but weakened the expression of the disease-defense gene, chia5. TIBA also decreased the expression of hsr203J but increased the expression of chia5. Thus, the auxin can reverse harpin(Xoo)-induced HR.

Use of fluorescence-activated vesicle sorting for isolation of Naked2-associated, basolaterally targeted exocytic vesicles for proteomics analysis.

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFalpha-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na(+)/K(+)-ATPase alpha1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.

Investigation of physiological and molecular mechanisms conferring diurnal variation in auxinic herbicide efficacy.

The efficacy of auxinic herbicides, a valuable weed control tool for growers worldwide, has been shown to vary with the time of day in which applications are made. However, little is known about the mechanisms causing this phenomenon. Investigating the differential in planta behavior of these herbicides across different times of application may grant an ability to advise which properties of auxinic herbicides are desirable when applications must be made around the clock. Radiolabeled herbicide experiments demonstrated a likely increase in ATP-binding cassette subfamily B (ABCB)-mediated 2,4-D and dicamba transport in Palmer amaranth (Amaranthus palmeri S. Watson) at simulated dawn compared to mid-day, as dose response models indicated that many orders of magnitude higher concentrations of N-1-naphthylphthalamic acid (NPA) and verapamil, respectively, are required to inhibit translocation by 50% at simulated sunrise compared to mid-day. Gas chromatographic analysis displayed that ethylene evolution in A. palmeri was higher when dicamba was applied during mid-day compared to sunrise. Furthermore, it was found that inhibition of translocation via 2,3,5-triiodobenzoic acid (TIBA) resulted in an increased amount of 2,4-D-induced ethylene evolution at sunrise, and the inhibition of dicamba translocation via NPA reversed the difference in ethylene evolution across time of application. Dawn applications of these herbicides were associated with increased expression of a putative 9-cis-epoxycarotenoid dioxygenase biosynthesis gene NCED1, while there was a notable lack of trends observed across times of day and across herbicides with ACS1, encoding 1-aminocyclopropane-1-carboxylic acid synthase. Overall, this research indicates that translocation is differentially regulated via specific protein-level mechanisms across times of application, and that ethylene release, a chief phytotoxic process involved in the response to auxinic herbicides, is related to translocation. Furthermore, transcriptional regulation of abscisic acid involvement in phytotoxicity and/or translocation are suggested.

A magic triangle for experimental phasing of macromolecules.

Obtaining phase information for the solution of macromolecular structures is still one of the bottlenecks in X-ray crystallography. 5-Amino-2,4,6-triiodoisophthalic acid (I3C), in which three covalently bound iodines form an equilateral triangle, was incorporated into proteins in order to obtain phases by single-wavelength anomalous dispersion (SAD). An improved binding capability compared with simple heavy-metal ions, ready availability, improved recognition of potential heavy-atom sites and low toxicity make I3C particularly suitable for experimental phasing.

Structure determination of the cancer-associated Mycoplasma hyorhinis protein Mh-p37.

The crystal structure of the Mycoplasma hyorhinis protein Mh-p37 has been solved and refined to 1.9 A resolution. This is the first de novo structure to be determined using the recently described heavy-atom reagent [Beck et al. (2008), Acta Cryst. D64, 1179-1182] 5-amino-2,4,6-triiodoisophthalic acid (I3C), which contains three I atoms arranged in an equilateral triangle, by SIRAS methods. Data collection was performed in-house at room temperature. SHELXD and SHELXE were used to determine the I-atom positions and phase the native protein and PHENIX AutoBuild software was used to automatically fit the amino-acid sequence to the electron-density map. The structure was refined using SHELX97 to an R(cryst) of 18.6% and an R(free) of 24.0%. Mh-p37 is an alpha/beta protein with two well defined domains which are separated by a deep cleft. An unanticipated ligand bound in the center of the molecule at the base of the cleft has been modeled as thiamine pyrophosphate or vitamin B(1). Retrospective attempts to solve the crystal structure by Patterson search methods using either isomorphous or anomalous differences failed. Additionally, attempts to use proteins with the highest structural homology in the Protein Data Bank to phase the data by molecular replacement were unsuccessful, most likely in hindsight because of their poor structural agreement. Therefore, the I3C reagent offers an alternative, quick and inexpensive method for in-house phasing of de novo structures where other methods may not be successful.

The mineralization of 5-amino-2,4,6-triiodoisophthalic acid by a two-stage fixed-bed reactor.

Iodinated X-ray contrast media have been detected in hospital effluent, sewage treatment plant effluent, rivers and groundwater aquifers. No process has been developed to remove triiodinated aromatic molecules. In this paper, we present a biological sequential process using an anaerobic fixed-bed reactor coupled in series with an aerobic fixed-bed reactor for degrading 5-amino-2,4,6-triiodoisophthalic acid (ATIA), the core structure of a X-ray contrast media family. The results obtained showed that the coupled reactor eliminated up to 870+/-44 mg of carbon L(-1) day(-1), with a molar ethanol/ATIA ratio of 4 in the feeding medium. The anaerobic reactor (ANR) undertook the majority of the deiodination of the aromatic nucleus and had a maximum deiodination rate of 23.4+/-0.06 mM day(-1). The aerobic reactor (AER) mineralized ATIA and was also able to eliminate its metabolites. This study suggests that the mineralization of ATIA can be achieved efficiently in a coupled anaerobic-aerobic bioreactor.

Tankyrase recruitment to the lateral membrane in polarized epithelial cells: regulation by cell-cell contact and protein poly(ADP-ribosyl)ation.

PARsylation [poly(ADP-ribosyl)ation] of proteins is implicated in the regulation of diverse physiological processes. Tankyrase is a molecular scaffold with this catalytic activity and has been proposed as a regulator of vesicular trafficking on the basis, in part, of its Golgi localization in non-polarized cells. Little is known about tankyrase localization in polarized epithelial cells. Using MDCK (Madin-Darby canine kidney) cells as a model, we found that E-cadherin-mediated intercellular adhesion recruits tankyrase from the cytoplasm to the lateral membrane (including the tight junction), where it stably associates with detergent-insoluble structures. This recruitment is mostly completed within 8 h of calcium-induced formation of cell-cell contact. Conversely, when intercellular adhesion is disrupted by calcium deprivation, tankyrase returns from the lateral membrane to the cytoplasm and becomes more soluble in detergents. The PARsylating activity of tankyrase promotes its dissociation from the lateral membrane as well as its ubiquitination and proteasome-mediated degradation, resulting in an apparent protein half-life of approximately 2 h. Inhibition of tankyrase autoPARsylation using H2O2-induced NAD+ depletion or PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide hydrochloride] treatment results in tankyrase stabilization and accumulation at the lateral membrane. By contrast, stabilization through proteasome inhibition results in tankyrase accumulation in the cytoplasm. These data suggest that cell-cell contact promotes tankyrase association with the lateral membrane, whereas PARsylating activity promotes translocation to the cytosol, which is followed by ubiquitination and proteasome-mediated degradation. Since the lateral membrane is a sorting station that ensures domain-specific delivery of basolateral membrane proteins, the regulated tankyrase recruitment to this site is consistent with a role in polarized protein targeting in epithelial cells.

Auxin distribution and transport during embryogenesis and seed germination of Arabidopsis.

Auxin distribution during embryogenesis and seed germination were studied with transgenic Arabidopsis plants expressing GUS gene driven by a synthetic DR5 promoter, an auxin responsive promoter. The results showed that GUS activity is higher in ends of hypophysis and cotyledon primordia of heart-, torpedo- and cotyledon-stage embryos, leaf tip area, lateral root primordia, root apex and cotyledon of young seedlings. And GUS accumulated in root apex of the seedlings grown on auxin transport inhibitor containing media. All these suggested that above-mentioned part of the organs and tissues have a higher level of auxin, and auxin polar transport inhibitor could cause the accumulation of auxin in root apex. And auxin transport inhibitor also resulted in aberration of Arabidopsis leaf pattern formation, root gravitropism and elongation.

Cholangiographic excretion studies: a comparison of iodipamide and iodoxamate in the dog.

Iodoxamic acid is a new hexaiodinated cholegraphic contrast agent. The methylglucamine salts of iodoxamate and iodipamide were administered to labrador dogs as an intravenous infusion. Bile salts were also infused. The biliary concentration and output of the two agents were compared. Bile flow rate, bile salt concentration and bile salt output with the two agents were also compared. The biliary output of iodoxamate (0.70-0.78 mumol/min/kg) was more than 50% higher than the iodipamide output (0.46 mumol/min/kg). Bile salt output and concentration with iodoxamate infusion were lower than with iodipamide infusion. The bile flow rate was higher with the new agent. The complementary effects of increased contrast output and decreased bile salt output with the new agent led to a significantly higher biliary iodine concentration compared with iodipamide. The results of this study support the suggestion that iodoxamate represents a significant advance in the cholegraphic contrast media field.

Biotransformation of ioglycamic acid, iodoxamic acid and iotroxic acid in man.

The biotransformation of the 131I-labeled cholegraphic media ioglycamic acid, iodoxamic acid and iotroxic acid in man is investigated. Plasma, urine and fistular bile were analyzed for unchanged and metabolized constituents of the administered substances using thin layer chromatography. No metabolites were found in plasma, but up to two were found in urine in addition to unchanged contrast media (a total of 50% of the total elimination in 24 hr. urine). A metabolite was only found in the fistular bile after the injection of iotroxic acid.

The biliary and urinary excretion and the choleretic effect of ioglycamide in dogs.

The biliary and urinary excretion and the choleretic effect of ioglycamide were studied in unanesthetized bile fistula dogs using stepwise increasing infusion rates to obtain multiple steady states. The results are compared with data from previously reported experiments in the same animals using iodoxamate and iodipamide. The rate of biliary excretion and the choleretic effect of ioglycamide are similar to those of iodipamide and iodoxamate. Like iodipamide and iodoxamate, the relation between infusion rate or plasma concentration and biliary excretion or concentration of ioglycamide are hyperbolic and can be fitted to saturation kinetics. Quantitatively, the excretion of ioglycamide and iodipamide are virtually identical. However, for any equimolar infusion rate or plasma concentration, more iodoxamate than ioglycamide is excreted in the bile. Despite the greater biliary excretion of iodoxamate, the maximum biliary concentration of ioglycamide, iodipamide, and iodoxamate is the same at low basal bile flow because the choleretic effects of the three compounds are equal. The data suggest that, theoretically, with any equimolar dose ioglycamide will be identical to iodipamide as a contrast material for intravenous cholangiography, but that iodoxamate may be superior to ioglycamide because more iodoxamate is excreted in the bile. This advantage of iodoxamate might become apparent clinically in patients with high basal bile flow or if smaller doses of the contrast material are used. However, at the presently recommended doses of the two compounds, it is unlikely that the use of ioglycamide for intravenous cholangiography will be any different than iodoxamate.

Characterization of liposomes containing iodine-125-labeled radiographic contrast agents.

Multilamellar liposomes were prepared containing either iodine-125-labeled (125I) diatrizoate or 125-I labeled iotrol in their aqueous phase. The in vitro permeabilities of liposomes containing both contrast agents were measured in the presence of saline and serum at 37 degrees C. Two different phospholipid compositions were studied: phosphatidylcholine/cholesterol/stearylamine (PC/C/S, 8: 1:1 molar ratio) and distearoylphosphatidylcholine/sphingomyelin (DSPC/SM, 5:2 mole ratio). In saline, similar permeabilities were observed for the four phospholipid-contrast agent combinations. In serum, however, leakage of 125I activity was 2 to 3 times greater from PC/C/S liposomes than from vesicles composed of DSPC/SM. When PC/C/S liposomes that contained 125I-diatrizoate were injected into rats, the clearance half-times for 125I activity from the liver, spleen, and whole body were 4.4 hours, 4.5 hours, and 2.8 hours, respectively. Liposomes composed of DSPC/SM cleared at a significantly slower rate from the liver, spleen, and whole body with half-times of 24.0 hours, 18.4 hours, and 17.2 hours observed from these tissues, respectively.

Gastrointestinal radiology. Pharmacokinetics of iopanoate and iodoxamate in rhesus monkeys.

The pharmacokinetics, biliary excretion, plasma protein binding, enterohepatic circulation, and biotransformation of iopanoic acid and iodoxamic acid in the rhesus monkey were evaluated by a dynamic infusion method. The dynamic method has the advantage that the pharmacokinetic parameters involved in the hepatic uptake and biliary excretion can be evaluated from a single infusion experiment. The percentage of iodoxamic acid not bound to plasma protein varied from 6.1-41.2% as iodoxamic acid plasma levels were from 42 microM to 912 microM. Using the Freundlich isotherm approach, more than one class of binding site for iodoxamic acid was found. A saturable biliary excretion mechanism or hepatic uptake mechanism was determined with a Vmax of 1.03 microM/kg/min. Less than 1% of iodoxamic acid injected into the duodenum was recovered in the bile in 12 hours. Iodoxamic acid was found to exist in blood as an unchanged species. Iopanoic acid was extremely highly bound to monkey plasma protein. As blood concentration increased from 18.9 to 464 microM, the percentage unbound in plasma protein varied from 0.1-2.8%. Biliary excretion rates of iopanoic acid were fitted by a computer to the Michaelis-Menten equation against unbound plasma concentration and the average Vmax value was found to be 0.85 microM/kg/min with an average Kmax value of 0.253. Iopanoic acid was found to exist in monkey blood as unchanged species and in the bile mainly as an ester glucuronide. Coadministration experiments revealed that the interaction of iodoxamic acid and iopanoic acid in the monkey is complex. The compounds appear to compete for plasma protein binding sites as well as for binding sites on intrahepatic protein. The biliary excretion data seem to fit the ligant exclusion model, in which iopanoic acid acts as an inhibitor and competes with iodoxamic acid for binding to either of two identical sites in the liver, which, presumably, is the rate-limiting step in the liver's overall elimination of these radiographic agents.

Biliary excretion of iodipamide and iodoxamate in normal and common bile duct-obstructed dogs.

Iodipamide and iodoxamate were compared at equimolar clinical dosages in dogs with normal, incompletely obstructed and completely obstructed common bile ducts. Forty-eight experiments were performed under general anesthesia in six cholecystomized chronic bile fistula dogs. The peak biliary iodoxamate excretion rate, but not the peak bile iodoxamate concentration, was significantly higher with normal and incompletely obstructed common bile ducts. In complete obstruction, both a significantly higher total biliary iodoxamate excretion and concentration were obtained, but this was still insufficient for radiographic opacification by conventional technique. Lesser toxicity of iodoxamate is suggested by its significantly lower serum levels, its higher bile: urine excretion ratio and its faster compensatory urinary excretion in complete common bile duct obstruction. Iodoxamate appears on this evidence to be a better cholangiographic contrast agent than iodipamide.

Penetration of subarachnoid contrast medium into rabbit spinal cord. Comparison between metrizamide and iohexol.

The penetration into rabbit spinal cord of two nonionic contrast media, iohexol and metrizamide, and a reference tracer, technetium DTPA, were compared. The spinal subarachnoid space was perfused for 4 hours with a CSF solution to which technetium DTPA and either iohexol or metrizamide had been added. The contrast media and technetium DTPA concentrations reached a plateau level in CSF outflow within 80 minutes. The contrast media concentrations in CSF were higher than the technetium DTPA (P less than .001). In the cord tissue, technetium DTPA reached higher concentrations than the contrast media (P less than .001), and iohexol reached higher concentrations relative to technetium DTPA than metrizamide (P less than .001). The mean contrast media distribution volumes in the thoracic cord were 13% (iohexol) and 12% (metrizamide). The smaller distribution volume observed for metrizamide could be related to the larger effective size of "associated" metrizamide molecules or an interference with diffusion perhaps related to binding to glucose carriers.

Human pharmacokinetics of iohexol. A new nonionic contrast medium.

The pharmacokinetics of iohexol, a new nonionic, water-soluble contrast medium, have been determined after intravenous injection in 20 healthy volunteers, at four different dose levels (125-500 mg I/kg). The apparent volume of distribution was 0.27 1/kg, indicating distribution in the extracellular water. The biologic half-life was 121 minutes, comparable with that of other intravascular contrast media. Iohexol was excreted completely unmetabolized in the urine, with a 100% recovery 24 hours after injection. A comparison of iohexol and chromium-51 (51Cr)-EDTA clearances indicates that iohexol is mainly excreted by glomerular filtration. The 51Cr-EDTA clearance was the same when injected separately and concomitantly with iohexol, indicating that glomerular filtration rate is not affected by iohexol. No dose dependency was observed in the investigated parameters t1/2 alpha, t1/2 beta, Vd, ClT or ClR. Iohexol pharmacokinetics are in correspondence with previously reported data on intravascular contrast media.

Biliary excretion of iodipamide and iodoxamate in dogs with hepatic dysfunction induced by oral administration of dimethylnitrosamine.

Iodipamide and iodoxamate were compared in equimolar clinical dosages in five cholecystectomized chronic bile fistula dogs in which hepatic dysfunction was produced by oral administration of a total dose of 480 and 960 microliters dimethylnitrosamine (DMNA), respectively. After both DMNA dosages, the peak biliary excretion rate for iodoxamate was significantly higher than for iodipamide (p less than 0.01). The peak bile iodine concentration was not significantly different for the two agents (480 microliter DMNA: p less than 0.1; 960 microliter DMNA: p = 0.07). On the basis of this investigation, it is suggested that iodoxamate should not significantly improve the opacification of the biliary system in patients with hepatic dysfunction.

Mechanism of renal excretion of various X-ray contrast materials in rabbits.

The excretory behavior of nine nephrotropic contrast agents with varying physicochemical properties such as charge, lipophilicity, and molecular size was investigated. Renal clearance in comparison with inulin was determined by means of the continuous infusion method. Each contrast agent was infused at three dose levels in four to six rabbits. The investigations show that tubular transportation in proportion to glomerular filtration decreases with increasing dosages of all the contrast agents. Thus, with the highest concentration in plasma all contrast agents are eliminated at more or less the glomerular filtration rate (GFR). After administration of the low dosages the following differences are found: 1) Net tubular secretion increases for the monomeric contrast agent acids with increasing lipophilicity, in the order diatrizoate congruent to iothalamate less than iodamide less than acetrizoate. 2) The clearance studies do not reveal any tubular secretion or reabsorption for a hydrophilic cationic contrast agent. 3) The nonionic contrast agents do not show net secretion. The more lipophilic they are, the more they are reabsorbed. 4) Two dimeric contrast agents also do not reveal any tubular secretion. They seem to be reabsorbed more than monomers with the same charge.