Elimination kinetic of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate ester metabolites in calves' urine

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C-18 SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CC alpha) for steroids were below 0.1 mu g L-1, except for 19-norandrosterone (CC alpha = 0.7 mu g L-1) and estrone (CC alpha = 0.3 mu g L-1). Kinetics of elimination of the administered 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate were established by monitoring 17 beta-estradiol, 17 alpha-estradiol, estrone and 17 beta-nandrolone, 17 alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17 alpha-estradiol and 17 alpha-nandrolone (> 20 and 2 mg L-1, respectively after 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate administration) whereas 17 beta-estradiol, estrone, 17 beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the mu g L-1 level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17 beta form. (c) 2008 Elsevier Ltd. All rights reserved.

Development and validation of dry reagent time-resolved fluoroimmuno assays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 961 23\EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin a-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TR-FIAs, the results being available within 1 h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.

INFLUENCE OF ULTRASOUND ON THE PERCUTANEOUS-ABSORPTION OF NICOTINATE ESTERS

The influence of ultrasound on the percutaneous absorption of three nicotinate esters was investigated in 10 healthy volunteers in a double-blind placebo controlled crossover clinical trial. Using a specially designed experimental protocol, the effect of continuous output ultrasound (at frequency 3.0 MHz and intensity 1.0 W/cm2 for 5 min) on the percutaneous absorption of methyl, ethyl, and hexyl nicotinates, from gel bases, was investigated. A placebo control, involving massage with each of the gels, without ultrasound for 5 min, was also incorporated. The pharmacodynamic parameter of vasodilation caused by the nicotinates was used to monitor the percutaneous absorption of the drugs. Laser Doppler velocimetry, a noninvasive optical technique, was used to measure vasodilation of the cutaneous vessels within the treatment site. Ultrasound treatment led to enhanced vasodilator response to the nicotinates, therefore indicating an enhancement of their percutaneous absorption. These agents may prove to be useful compounds in examination of the mechanism of action of phonophoresis.

Genomic instability in Chinese hamster cells after exposure to X rays or alpha particles of different mean linear energy transfer

Evidence has accumulated that radiation induces a transmissible persistent destabilization of the genome, which mag. result in effects arising in the progeny of irradiated but surviving cells. An enhanced death rate among the progeny of cells surviving irradiation persists for many generations in the form of a reduced plating efficiency. Such delayed reproductive death is correlated with an increased occurrence of micronuclei. Since it has been suggested that radiation-induced chromosomal instability might depend on the radiation quality, we investigated the effects of alpha particles of different LET by looking at the frequency of delayed micronuclei in Chinese hamster V79 cells after cytochalasin-induced block of cell division, A dose-dependent increase in the frequency of micronuclei was found in cells assayed 1 week postirradiation or later. Also, there was a persistent increase in the frequency of dicentrics in surviving irradiated cells, Moreover, we found an increased micronucleus frequency in all of the 30 clones isolated from individual cells which had been irradiated with doses equivalent to either one, two or three alpha-particle traversals per cell nucleus, We conclude that the target for genomic instability in Chinese hamster cells must be larger than the cell nucleus. (C) 1997 by Radiation Research Society

DNA double-strand break distributions in X-ray and alpha-particle irradiated V79 cells: Evidence for non-random breakage

Many studies have shown that with increasing LET of ionizing radiation the RBE (relative biological effectiveness) for dsb (double strand breaks) induction remains around 1.0 despite the increase in the RBE for cell killing. This has been attributed to an increase in the complexity of lesions, classified as dsb with current techniques, at multiply damaged sites. This study determines the molecular weight distributions of DNA from Chinese hamster V79 cells irradiated with X-rays or 110 keV/mu m alpha-particles. Two running conditions for pulsed-field gel-electrophoresis were chosen to give optimal separation of fragments either in the 225 kbp-5.7 Mbp range or the 0.3 kbp to 225 kbp range. Taking the total fraction of DNA migrating into the gel as a measure of fragmentation, the RBE for dsb induction was less than 1.0 for both molecular weight regions studied. The total yields of dsb were 8.2 x 10(-9) dsb/Gy/bp for X-rays and 7.8 x 10(-9) dsb/Gy/bp for a-particles, measured using a random breakage model. Analysis of the RBE of alpha-particles versus molecular weight gave a different response. In the 0.4 Mbp-57 Mbp region the RBE was less than 1.0; however, below 0.4 Mbp the RBE increased above 1.0. The frequency distributions of fragment sizes were found to differ from those predicted by a model assuming random breakage along the length of the DNA and the differences were greater for alpha-particles than for X-rays. An excess of fragments induced by a single-hit mechanism was found in the 8-300 kbp region and for X-rays and alpha-particles these corresponded to an extra 0.8 x 10(-9) and 3.4 x 10(-9) dsb/bp/Gy, respectively. Thus for every alpha-particle track that induces a dsb there is a 44% probability of inducing a second break within 300 kbp and for electron tracks the probability is 10%. This study shows that the distribution of damage from a high LET alpha-particle track is significantly different from that observed with low LET X-rays. In particular, it suggests that the fragmentation patterns of irradiated DNA may be related to the higher-order chromatin repealing structures found in intact cells.

A study of endonuclease III-sensitive sites in irradiated DNA: detection of alpha-particle-induced oxidative damage

An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA, Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality. With increasing linear energy transfer, it is predicted that complex lesions will be formed within 1-20 bp regions of the DNA, As well as strand breaks, these sites may contain multiple damaged bases, We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks. Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH radicals with either Co-60 gamma-rays or alpha-particles from a Pu-238 source. Under low scavenging conditions (10 mM Tris) gamma-irradiation induced 7.1x10(-7) ssb Gy/bp, which increased 3.7-fold to 2.6 x 10(-6) ssb Gy/bp with endo III treatment. In contrast the yields of dsb increased by 4.2-fold from 1.5 x 10(-8) to 6.3 x 10(-8) dsb Gy/bp, This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment. For alpha-particles this increased to 9%. Given that endo III sensitive sites may only constitute similar to 40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in alpha-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity.

Delayed lethality, apoptosis and micronucleus formation in human fibroblasts irradiated with X-rays or alpha-particles

Purpose: To determine the yields of cell lethality and micronucleus formation measured immediately after irradiation or at delayed times in primary human fibroblasts exposed to X-rays or alpha-particles.

The role of higher-order chromatin structure in the yield and distribution of DNA double-strand breaks in cells irradiated with X-rays or alpha-particles

Purpose: To determine whether the non-random distributions of DNA double-strand breaks in cells observed after alpha-particle irradiation are related to the higher-order structure of the chromatin within the nucleus.