Evolution of E. coli on [U-13C]Glucose Reveals a Negligible Isotopic Influence on Metabolism and Physiology.

13C-Metabolic flux analysis (13C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled 13C-glucose, a commonly used isotope for 13C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (13C) or unlabeled (12C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between 12C and 13C-glucose as a carbon source for E. coli growth.

Monte Carlo simulations of the relative biological effectiveness for DNA double strand breaks from 300 MeV u(-1) carbon-ion beams.

Monte Carlo simulations are used to calculate the relative biological effectiveness (RBE) of 300 MeV u(-1) carbon-ion beams at different depths in a cylindrical water phantom of 10 cm radius and 30 cm long. RBE values for the induction of DNA double strand breaks (DSB), a biological endpoint closely related to cell inactivation, are estimated for monoenergetic and energy-modulated carbon ion beams. Individual contributions to the RBE from primary ions and secondary nuclear fragments are simulated separately. These simulations are based on a multi-scale modelling approach by first applying the FLUKA (version 2011.2.17) transport code to estimate the absorbed doses and fluence energy spectra, then using the MCDS (version 3.10A) damage code for DSB yields. The approach is efficient since it separates the non-stochastic dosimetry problem from the stochastic DNA damage problem. The MCDS code predicts the major trends of the DSB yields from detailed track structure simulations. It is found that, as depth is increasing, RBE values increase slowly from the entrance depth to the plateau region and change substantially in the Bragg peak region. RBE values reach their maxima at the distal edge of the Bragg peak. Beyond this edge, contributions to RBE are entirely from nuclear fragments. Maximum RBE values at the distal edges of the Bragg peak and the spread-out Bragg peak are, respectively, 3.0 and 2.8. The present approach has the flexibility to weight RBE contributions from different DSB classes, i.e. DSB0, DSB+ and DSB++.

Degradation of the persistent organic pollutant [14C]heptachlor in Japanese field soils.

The fate of [(14)C]heptachlor in Saitama soil and the degradation of [(14)C]heptachlor in four Japanese field soils over 112 d after application were investigated. Heptachlor was degraded mainly to cis-heptachlor epoxide by a biotic process and to 1-hydroxychlordene by an abiotic process in the field soils. Volatilization of heptachlor and cis-heptachlor epoxide from the soil was observed over the experimental period. The amount of 1-hydroxychlordene produced in the soils appeared to be related to the soil water contents. Because heptachlor and heptachlor epoxides are predicted to volatilize to the atmosphere and to persist in soils, these compounds are thought to spread among Japanese environmental compartments even after a ban on their use.

Do Chernobyl-like contaminations with (137)Cs and (90)Sr affect the microbial community, the fungal biomass and the composition of soil organic matter in soil?

(137)Cs and (90)Sr are the main radionuclides responsible for contamination of agricultural soils due to core melts in nuclear power plants such as Chernobyl or Fukushima. The present study focused on effects of Chernobyl-like contaminations on the bacterial and fungal community structure, the fungal biomass and the formation of soil organic matter in native and in sterilized and reinoculated soils. 2% wheat straw [m/m] was applied to a typical agricultural soil, artificially contaminated with (137)Cs and (90)Sr, and it was then incubated in microcosms for three months at 20 °C and 50% of the water-holding capacity. The development of the microbial communities was monitored with 16S and 18S rDNA denaturing gradient gel electrophoresis (DGGE). The quantification of the ergosterol content was used as a proxy for changes in the fungal biomass. Changes in the soil organic matter were determined using the (13)C cross polarization/magic angle spinning nuclear magnet resonance technique ((13)C-CP/MAS NMR). Slight but significant population shifts in the DGGE gel patterns could be related to the applied radionuclides. However, radiation-induced impacts could not be seen in either the chemical composition of the soil organic matter or in the development of the fungal biomass. Impacts caused by sterilization and reinoculation prevailed in the microcosms of the present study. Contaminations with (137)Cs or (90)Sr up to 50-fold that of the hotspots occurring in Chernobyl led to minor changes in soil microbial functions suggesting a strong resilience of natural soils with respect to radioactive contamination.

Cytochrome P450 11A1 bioactivation of a kinase inhibitor in rats: use of radioprofiling, modulation of metabolism, and adrenocortical cell lines to evaluate adrenal toxicity.

A drug candidate, BMS-A ((N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl) 2-oxo-1,2-dihydropyridine- 3-carboxamide)), was associated with dose- and time-dependent vacuolar degeneration and necrosis of the adrenal cortex following oral administration to rats. Pretreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, ameliorated the toxicity. In vivo and in vitro systems, including adrenal cortex-derived cell lines, were used to study the mechanism responsible for the observed toxicity. Following an oral dose of the C-14 labeled compound, two hydroxylated metabolites of the parent (M2 and M3) were identified as prominent species found only in adrenal glands and testes, two steroidogenic organs. In addition, a high level of radioactivity was covalently bound to adrenal tissue proteins, 40% of which was localized in the mitochondrial fraction. ABT pretreatment reduced localization of radioactivity in the adrenal gland. Low levels of radioactivity bound to proteins were also observed in testes. Both M3 and covalent binding to proteins were found in incubations with mitochondrial fraction isolated from adrenal tissue in the presence of NADPH. In vitro formation of M3 and covalent binding to proteins were not affected by addition of GSH or a CYP11B1/2 inhibitor, metyrapone (MTY), but were inhibited by ketoconazole (KTZ) and a CYP11A1 inhibitor, R-(+)-aminoglutethimide (R-AGT). BMS-A induced apoptosis in a mouse adrenocortical cell line (Y-1) but not in a human cell line (H295R). Metabolite M3 and covalent binding to proteins were also produced in Y-1 and to a lesser extent in H295R cells. The cell toxicity, formation of M3, and covalent binding to proteins were all diminished by R-AGT but not by MTY. These results are consistent with a CYP11A1-mediated bioactivation to generate a reactive species, covalent binding to proteins, and subsequently rat adrenal toxicity. The thorough understanding of the metabolism-dependent adrenal toxicity was useful to evaluate cross-species adrenal toxicity potential of this compound and related analogues.

Photochemical analysis of 14C-fenhexamid in aqueous solution and structural elucidation of a new metabolite.

The photodegradation kinetics and the break down pathway of fenhexamid were studied in aqueous systems using [phenyl-UL-14C]- and [carbonyl-14C]-labelled compounds. The photolysis of fenhexamid followed first-order kinetics. The degradation rate of fenhexamid was significantly influenced by the solution pH with rate constants (k) of 2.11×10(-2), 4.47×10(-2), 6.11×10(-1) and 1.69 h(-1) at pH 5.0, 6.6, 7.3 and 9.0, respectively. Fenhexamid exhibited no significant change in degradation rate in the presence of acetone and hydrogen peroxide, while humic and fulvic acids retarded the degradation rate, because they shielded the active molecules from light. However, in phosphate medium, the photolysis rate was significantly enhanced as a function of concentration. About 3-8% and 10-25% photo mineralization were observed, using [carbonyl-14C]- and [phenyl-UL-14C]-labelled fenhexamid in aqueous solutions at different pH, respectively. In addition to four known metabolites, one major and five minor photoproducts out of which one is reported for the first time, were identified using high resolution LC-MS/MS and NMR. The toxicity of the new metabolite was tested against the fish Oncorhynchus mykiss with no lethal effect at 100 mg L(-1).

Single extreme low dose/low dose rate irradiation causes alteration in lifespan and genome instability in primary human cells.

To investigate the long-term biological effect of extreme low dose ionising radiation, we irradiated normal human fibroblasts (HFLIII) with carbon ions (290 MeV u(-1), 70 keV microm(-1)) and gamma-rays at 1 mGy (total dose) once at a low dose rate (1 mGy 6-8 h(-1)), and observed the cell growth kinetics up to 5 months by continuous culturing. The growth of carbon-irradiated cells started to slow down considerably sooner than that of non-irradiated cells before reaching senescence. In contrast, cells irradiated with gamma-rays under similar conditions did not show significant deviation from the non-irradiated cells. A DNA double strand break (DSB) marker, gamma-H2AX foci, and a DSB repair marker, phosphorylated DNA-PKcs foci, increased in number when non-irradiated cells reached several passages before senescence. A single low dose/low dose rate carbon ion exposure further raised the numbers of these markers. Furthermore, the numbers of foci for these two markers were significantly reduced after the cells became fully senescent. Our results indicate that high linear energy transfer (LET) radiation (carbon ions) causes different effects than low LET radiation (gamma-rays) even at very low doses and that a single low dose of heavy ion irradiation can affect the stability of the genome many generations after irradiation.

Bioaccumulation of 51Cr, 63Ni and 14C in Baltic Sea benthos.

The Baltic Sea is a species-poor, semi-enclosed, brackish sea, whose sediments contain a wide range of contaminants, including sediment-associated metals and radionuclides. In this study, we have examined and compared bioaccumulation kinetics and assimilation efficiencies of sediment-associated (51)Cr, (63)Ni and (14)C in three key benthic invertebrates (the deposit-feeding Monoporeia affinis, the facultative deposit-feeding Macoma baltica, and the omnivorous Halicryptus spinulosus). Our results demonstrate that (i) all radionuclides were accumulated, (ii) the different radionuclides were accumulated to various extents, (iii) small changes in organic carbon concentration can influence the accumulation, and (iv) the degree of accumulation differed only slightly between species. These processes, together with sediment resuspension and bioturbation, may remobilise trace metals from the sediment to the water and to higher trophic levels, and therefore should be taken into account in exposure models and ERAs.

[Carbon-11 labeled diacylglycerol for signal transduction imaging by positron CT: evaluation of the quality and safety for clinical use].

To elucidate the synaptic transmission in the neural system, we have been developing fundamental studies for intracellular signaling. For clinical application of carbon-11 labeled diacylglycerol (1-[1-11C]butyryl-2-palmitoyl-rac-glycerol: 11C-DAG) using positron emission computed tomography (PET), we evaluated the quality and the safety of 11C-DAG as the solution for injection. As a result, 11C-DAG was synthesized within 50 minutes, including the preparation step for injection. The half life time and energy spectrum of 11C-DAG were the same as the physical character of carbon-11, and other radioisotopes were not detected. In the quality control, 11C-DAG solution was negative in the examination of bacterial contamination and the pyrogen test in three successive synthesis procedures. In the acute toxicity test by administration of 11C-DAG and 100 mumol/kg of non-radioactive DAG to the rat intravenously, the systemic condition of the rat was not changed and no abnormalities were found in any organ 24 hours after administration. These findings indicated the safety of 11C-DAG solution. Clinical application of 11C-DAG using positron emission tomography may be useful to elucidate the dysfunction of intracellular signaling in disorders of higher cortical function such as Alzheimer disease.

Tumour development following internal exposures to radionuclides during the perinatal period.

Exposure to radiation from internally deposited radionuclides during the prenatal and/or neonatal periods bears a distinct oncogenic potential. The fundamental mechanisms of perinatal radionuclide carcinogenesis seem to be generally similar to those that pertain to external radiation exposures and other carcinogenic agents, but unique interactions may be superimposed. Specific dose-effect relationships differ among radionuclides; in many studies, there have been dose-related increases in the incidence of tumours or decreases in age at tumour appearance following prenatal or neonatal radiation exposure. Tumour incidences may be decreased, especially at high dose levels; these are usually attributable to cell death, inhibited development of target tissues or to endocrine malfunction. Age-related differences in predominant tumour types and/or sites of tumour development are often detected, and are explainable by the existence of nuclide-specific target organs or tissues, dosimetric factors and developmental considerations.

DNA strand breakage in Chinese hamster V79 cells caused by low levels of incorporated [3H] and [14C]thymidine.

A sensitive alkali-unwinding assay was used to measure DNA strand breakage in Chinese hamster V79 cells caused by low-level incorporation of methyl-labelled [3H] and [14C] thymidine, and to estimate the effective dose per disintegration relative to low doses of gamma-irradiation. Damage equivalent to 0.0035 +/- 0.0006 and 0.0014 +/- 0.0005 Gy was observed for each 3H and 14C disintegration respectively. These values agree well with those expected from the estimated nuclear radiation dose delivered by the beta particles if a relative biological effect (r.b.e.) of 1.0 is assumed, and suggest that strand-breakage produced by these isotopes is determined by the nuclear radiation dose delivered by the beta particles.