DNA profiling, telomere analysis and antioxidant properties as tools for monitoring ex situ seed longevity.

BACKGROUND AND AIMS: The germination test currently represents the most used method to assess seed viability in germplasm banks, despite the difficulties caused by the occurrence of seed dormancy. Furthermore, seed longevity can vary considerably across species and populations from different environments, and studies related to the eco-physiological processes underlying such variations are still limited in their depth. The aim of the present work was the identification of reliable molecular markers that might help in monitoring seed deterioration. METHODS: Dry seeds were subjected to artificial ageing and collected at different time points for molecular/biochemical analyses. DNA damage was measured using the RAPD (random amplified polymorphic DNA) approach while the seed antioxidant profile was obtained using both the DPPH (1,1-diphenyl, 2-picrylhydrazyl) assay and the Folin-Ciocalteu reagent method. Electron paramagnetic resonance (EPR) provided profiles of free radicals. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to assess the expression profiles of the antioxidant genes MT2 (type 2 metallothionein) and SOD (superoxide dismutase). A modified QRT-PCR protocol was used to determine telomere length. KEY RESULTS: The RAPD profiles highlighted different capacities of the two Silene species to overcome DNA damage induced by artificial ageing. The antioxidant profiles of dry and rehydrated seeds revealed that the high-altitude taxon Silene acaulis was characterized by a lower antioxidant specific activity. Significant upregulation of the MT2 and SOD genes was observed only in the rehydrated seeds of the low-altitude species. Rehydration resulted in telomere lengthening in both Silene species. CONCLUSIONS: Different seed viability markers have been selected for plant species showing inherent variation of seed longevity. RAPD analysis, quantification of redox activity of non-enzymatic antioxidant compounds and gene expression profiling provide deeper insights to study seed viability during storage. Telomere lengthening is a promising tool to discriminate between short- and long-lived species.

Reaction mechanisms in irradiated, precipitated, and mesoporous silica.

A matrix EPR spectroscopy study of the low temperature γ radiolysis of precipitated (Zeosil) and mesoporous high surface silica has afforded evidence of the formation of trapped H-atoms, H-atom centers, siloxy radicals ≡Si-O(•), anomalous silyl peroxy radicals ≡Si-OO(•) with reduced g tensor anisotropy, siloxy radical-cations (≡Si-O-Si≡)(+•), E' centers, and two species from Ge impurity. Coordination of peroxyl radicals with diamagnetic ≡Si(+) centers is proposed and tested by DFT computations in order to justify the observed g tensor. Coordination of H-atoms to ≡Si(+) centers is also proposed for the structure of the H-atom centers as an alternative model not requiring the intervention of Ge, Sn, or CO impurities. The DFT method has been employed to assess the electronic structure of siloxy radical-cations and its similarity with that of the carbon radical-cation analogues; the results have prompted a revision of the structures proposed in the literature for ST1 and ST2 centers. The comparison between the two types of silica has afforded evidence of different radiolysis mechanisms leading to a greater yield of trapped H-atoms and H-atom centers in zeosil silica, which is reckoned with the 4-fold greater concentration of silanol groups. Parallel radiolysis experiments carried out by using both types of silica with polybutadiene oligomers as adsorbate have afforded evidence of free valence and energy migration phenomena leading to irreversible linking of polybutadiene chains onto silica. Reaction mechanisms are proposed based on the detection of SiO2-bonded free radicals whose structure has been defined by EPR.

gamma-irradiation of PEGd,lPLA and PEG-PLGA multiblock copolymers: II. effect of oxygen and EPR investigation.

The purpose of this research was to evaluate how the presence of oxygen can affect irradiation-induced degradation reactions of PEGd,lPLA and PEG-PLGA multiblock copolymers submitted to gamma irradiation and to investigate the radiolytic behavior of the polymers. PEGd,lPLA, PEG-PLGA, PLA, and PLGA were irradiated by using a (60)Co irradiation source in air and under vacuum at 25 kGy total dose. Mw and Mn were evaluated by gel permeation chromatography. The stability study was carried out on three samples sets: (a) polymer samples irradiated and stored in air, (b) polymer samples irradiated and stored under vacuum, and (c) polymer samples irradiated under vacuum and stored in air. The thermal and radiolytic behavior was investigated by differential scanning calorimetry and electron paramagnetic resonance (EPR), respectively. Samples irradiated in air showed remarkable Mw and Mn reduction and Tg value reduction due to radiation-induced chain scission reactions. Higher stability was observed for samples irradiated and stored under vacuum. EPR spectra showed that the presence of PEG units in multiblock copolymer chains leads to: (a) decrease of the radiolytic yield of radicals and (b) decrease of the radical trapping efficiency and faster radical decay rates. It can be concluded that the presence of oxygen during the irradiation process and the storage phase significantly increases the entity of irradiation-induced damage.

Gamma irradiation effects on stability of poly(lactide-co-glycolide) microspheres containing clonazepam.

This work was aimed at evaluating the effects of gamma irradiation on the stability of microspheres made of a poly(lactide-co-glycolide) copolymer (PLGA) and loaded with 15% w/w of clonazepam (CLO). The influence of CLO on PLGA radiolysis mechanisms and the identification of possible irradiation markers were also investigated. Microspheres were prepared by means of a spray-drying method. gamma Irradiation was carried out either under vacuum or in air, at a dose of 25 kGy, by using a 60Co source. The stability of CLO loaded microspheres was evaluated over a 6-month period on the basis of drug content and dissolution profile. Radiolysis mechanisms were investigated by using electronic paramagnetic resonance (EPR) analysis. The microspheres irradiated under vacuum were stable over the considered period of time. After irradiation in air, CLO release rate increased by approximately 10%, and did not change further in the following period of storage. The EPR analysis showed some radicals arising from both the polymeric matrix and the active ingredient. Polymer/CLO spin transfer reactions suggest that CLO had a radio-stabilising effect on the polymeric matrix. In the loaded microspheres, the intensity in time of the CLO radical signal is sufficient for its possible use as irradiation marker.

Chemical and physical stability of hydroxypropylmethylcellulose matrices containing diltiazem hydrochloride after gamma irradiation.

In recent years, the exposure to gamma-radiation is an increasingly used method to sterilize or to reduce bacterial charge in drug-delivery devices. The aim of this study was to investigate whether the ionizing radiation may be responsible for drug inactivation or for the alteration of the functional excipient used to modulate drug release from a controlled-release delivery system. In this work, we investigated the physical and dissolution stability of prolonged release matrix tablets containing diltiazem hydrochloride, as a model drug, and hydroxypropylmethylcellulose (HPMC) of two different viscosity grades, as the retarding polymer, before and after exposure to increasing doses of gamma-rays. The results show that gamma-irradiation induces chemical modifications in the structure of the active agent, and also of the hydrophilic polymer. The electronic paramagnetic resonance analysis of gamma-irradiated diltiazem has afforded evidence of carbon radicals stemming from C-H bond ruptures and sulphur radicals, the latter being formed mainly after admission of air at room temperature. The major radical products in the HPMC polymer radiolysis have been reckoned with chain scission events in agreement with the results of viscosity measurements that show a progressive decrease of the average molecular weight with increasing the radiation dose. The elaboration of the viscosity data has led to linear relationships between the eta(o)/eta ratio and the radiation dose D which were rationalized with the following equation under the assumption of a Mark-Houwink Sakurada coefficient a approximately equal 1: eta(o)/eta = (1 + uy(o) D)(a). In this equation, D is gamma-radiation dose, eta(o) and eta are the reduced viscosities before and after the irradiation respectively, u is the number average degree of polymerization, and y(o) is the chain scission radiolytic yield. From the linear relationships G(chain scissions) = 1.2 x 10(-6) and 1.4 x 10(-6) moles/J have been obtained for the two HPMC polymer samples M100 and M4 of different molecular weight used in the experiments. These changes could be responsible for the alteration of the drug-release mechanism and reduced polymer efficacy in controlling drug release.

Polymers-gamma ray interaction. Effects of gamma irradiation on modified release drug delivery systems for oral administration.

The aim of this work is to verify the efficiency of two kinds of matrix tablets formulations containing PEO or PVA as retarding polymer. Moreover, since in the last years the exposure to ionizing radiation is a more and more used method to reduce bacterial charge in pharmaceutical products, the effects of gamma irradiation on these two kinds of polymers has been evaluated. The study is performed on matrix tablets containing diltiazem HCl, as model drug, and polyethylene oxides (PEO) of two different molecular weights or polyvinylalchool (PVA) of medium degree of hydrolysis, as drug release modulators. Dissolution of the matrices, release of diltiazem and morphological behaviour of the samples, before and after exposure to increasing doses of gamma irradiation, are investigated in order to verify their stability. The results show that the ionizing radiation does not modify significantly the dissolution trend of the PVA samples; on the contrary, the dissolution and the morphological behaviour of the PEO matrices is strongly affected by the radiation dose received. In particular, the dissolution rate of the irradiated PEO tablets dramatically increases as a function of the irradiation dose and the swelling process, which characterised the non-irradiated PEO samples, was replaced by a rapid erosion process responsible for the quickly dissolution of the matrices. The changes of the dissolution and morphological PEO tablets performances could be explained by a breaking of the polymeric chains (shown by EPR studies) as a consequence of the exposure to gamma rays. These chemical-structural modifications of the polymers are responsible for the reduced efficacy of the PEO systems in controlling the drug release rate.

Gamma irradiation effects and EPR investigation on poly(lactide-co-glycolide) microspheres containing bupivacaine.

The effects of gamma radiation on the stability of microspheres made of a polylactide-co-glycolide 50:50 copolymer (PLGA) and loaded with 40% bupivacaine (BU) were studied. The radiolysis mechanisms of BU and BU-loaded microspheres were investigated by using electronic paramagnetic resonance (EPR) analysis. Microspheres were prepared by means of a spray drying method. Gamma Irradiation was carried out in the open, at the dose of 25 kGy, by using a 60Co source. The stability of BU-loaded microspheres was evaluated over a 1-year period on the basis of drug content and dissolution profile. Non-irradiated microspheres were stable over the whole period under consideration. Immediately after irradiation the amount of BU released after 24 h from irradiated microspheres increased from 17 to 25%; in the following 3 months of storage it increased to about 35%, and then it kept constant for 1 year. Radicals generated by BU irradiation were identified by EPR analysis; the sensitivity to gamma radiation of BU was about four times lower than that of PLGA. Furthermore, the EPR spectra of loaded microspheres showed that the relative abundance of BU radicals plus PLGA radicals was proportionate to the electronic fractions of the components; this implies that no spin transfer BU/PLGA had occurred during gamma irradiation.

Neutron ESR dosimetry through ammonium tartrate with low Gd content.

This paper continues analyses on organic compounds for application in neutron dosimetry performed through electron spin resonance (ESR). Here, the authors present the results obtained by ESR measurements of a blend of ammonium tartrate dosemeters and gadolinium oxide (5 % by weight). The choice of low amount of Gd is due to the need of improving neutron sensitivity while not significantly influencing tissue equivalence. A study of the effect of gadolinium presence on tissue equivalence was carried out. The experiments show that the neutron sensitivity is enhanced by more than an order of magnitude even with this small additive content. Monte Carlo simulations on the increment of energy release due to gadolinium presence were carried, and the results were in good agreement with the experimental data.

EPR/alanine pellets with low Gd content for neutron dosimetry.

This paper reports on results obtained by electron paramagnetic resonance (EPR) measurements and Monte Carlo (MC) simulation on a blend of alanine added with low content of gadolinium oxide (5 % by weight) to improve the sensitivity to thermal neutron without excessively affecting tissue equivalence. The sensitivity is enhanced by this doping procedure of more an order of magnitude. The results are compared with those obtained with the addition of boric acid (50 % by weight) where boron is in its natural isotopic composition in order to produce low-cost EPR dosemeters. The gadolinium addition influences neutron sensitivity more than the boron addition. The presence of additives does not substantially change the fading of the EPR signal induced by neutrons. The MC simulations agree the experimental results in case of gadolinium addition.