Effect of oxygen concentration and selected protocol factors on viability and gene expression of mouse liver slices.

Precision cut liver slices (PCLSs) are widely used as a model to study hepatotoxicity. For culturing of PCLS diverse protocols are used which could affect slices viability and results. We aimed to identify the most optimal culture protocol for mouse PCLS. Slices were cultured for 24h under different concentrations of serum, glucose, insulin, and oxygen. Thereafter, slices viability was assessed by biochemical methods. Transcriptome analysis was performed to identify changes introduced by culture at different oxygen concentrations (20%, 40%, 60%, and 80% of oxygen). Medium composition did not affect the slices viability. Although metabolic competence was unaffected by oxygen concentrations, culturing at 80% of oxygen yielded slices with the best biochemical characteristics. The comparison of uncultured vs. cultured slices revealed 2524 genes to be differentially expressed. Genes involved in drug metabolism, peroxisomal and mitochondrial functions were down-regulated while several adaptive/stress response processes were up-regulated. Moreover, 80% of oxygen was the most favorable condition with respect to maintenance of expression of genes involved in drug and energy metabolism. The outcome of this study indicates that mouse PCLS are a valuable tool in research on hepatic functions and toxicity, particularly if they are cultured under a controlled oxygen concentration of 80%.

Bioassay based screening of steroid derivatives in animal feed and supplements.

Receptor binding transcription activation bioassays are valuable tools for the screening of steroid hormones in animal feed and supplements. However, steroid derivatives often lack affinity for their cognate receptor and do not show any direct hormonal activity by themselves. These compounds are thus not detected by these kinds of bioassays and need a bioactivation step in order to become active, both in vivo and in vitro. In this study a comparison was made between different in vitro activation methods for hormone esters and hormone glycosides. Testosterone acetate and testosterone decanoate were chosen as model compounds for the hormone esters, representing the broad range of steroid esters of varying polarities, while genistin was used as a substitute model for the steroid-glycosides. Concerning bioactivation of the steroids esters, the efficiency for alkaline hydrolysis was 90-100% and much better as compared to enzymatic deconjugation by esterase. As a result 1 µg testosterone ester per gram of animal feed could easily be detected by a yeast androgen bioassay. When comparing different enzyme fractions for deglycosilation, genistin was shown to be deconjugated most efficiently by ß-glucuronidase/aryl sulfatase from Helix pomatia, resulting in a significant increase of estrogenic activity as determined by a yeast estrogen bioassay. In conclusion, chemical and enzymatic deconjugation procedures for ester and glycoside conjugates respectively, resulted in a significant increase in hormonal activity as shown by the bioassay readouts and allowed effective screening of these derivatives in animal feed and feed supplements.

The ultimate veal calf reference experiment: hormone residue analysis data obtained by gas and liquid chromatography tandem mass spectrometry.

A lifetime controlled reference experiment has been performed using 42 veal calves, 21 males and 21 females which were fed and housed according to European regulations and common veterinary practice. During the experiment feed, water, urine and hair were sampled and feed intake and growth were monitored. Thus for the first time residue analysis data were obtained from guaranteed lifetime-untreated animals. The analysis was focused on the natural hormones estradiol and testosterone and their metabolites, on 17beta- and 17alpha-nortestosterone, on 17beta- and 17alpha-boldenone and androsta-1,4-diene-3,17-dione (ADD), and carried out by gas chromatography tandem mass spectrometry (GC/MS/MS), an estrogen bioassay and liquid chromatography (LC) MS/MS. Feed, water and hair samples were negative for the residues tested. Female calf urines showed occasionally low levels of 17alpha-estradiol and 17alpha-testosterone. On one particular sampling day male veal calf urines showed very high levels of 17alpha-testosterone (up to 1000 ng mL(-1)), accompanied by lower levels of estrone and 17beta-testosterone. Despite these extreme levels of natural testosterone, 17beta-boldenone was never detected in the same urine samples; even 17alpha-boldenone and ADD were only occasionally beyond CCalpha (maximum levels 2.7 ng mL(-1)). The data from this unique experiment provide a set of reference values for steroid hormones in calf urine and demonstrate that 17beta-boldenone is not a naturally occurring compound in urine samples.