Development and validation of a ready to use cryo-EROD assay for the standardized screening of dioxins and dioxin-like compounds in foodstuffs.

Recent European regulations have indicated the need for new bioanalytical screening methods capable of monitoring dioxin and dioxin-like compounds in foodstuffs and environmental samples, cost-effectively and with a quicker turnaround. Cryo-cells of the hepatic H4IIE line preserved in 96-well plates were exposed to sample extracts prepared from various foodstuffs and analysed for their content of dioxins and dioxin-like compounds by means of the 7-Ethoxyresorufin-O-Deethylase (EROD)-assay in two laboratories. Assay data were compared between both laboratories and results from instrumental analysis used as a confirmatory method. Additionally, cut-off values for the different studied matrices were derived. The current European regulation regarding methods of analysis for the control of foodstuffs was applied with the aim of determining the feasibility of the cryo-methodology. Results obtained in both laboratories were in congruence with the required validation parameters of the Commission Regulation (EU) No 2017/644. Cut-off values should be established matrix-dependent to reduce the rate of false compliant results and to keep the rate of false non-compliant results under control. In summary, the ready-to-use cryo-assay method for the bioanalytical screening of foodstuffs in control laboratories without cell-culture facilities has successfully proven to be accurate, far quicker and more cost effective than current methods.

Extraction of meat juices for isotopic analysis.

So far no standard procedure exists to obtain water of meat for isotopic (18)O/(16)O-water analysis. Fast extraction via heating the tissues is possible when considering certain boundary conditions. A specially designed vessel was tested with water and was then used for meat juice extraction. The reproducibility (σ) of δ(18)O-values was 0.12‰. Meat samples of six different species were analysed. Water of pork samples was extracted after open storage. Here, decreases in meat weight correspond to decreases in extract yield and to an increase in the (18)O/(16)O-ratio. The mean water contents in extracts was almost constant [93.2±0.05 wt% (p>0.05)]. The technique offers an opportunity to develop an automatic, mobile extraction device and to obtain extracts with no further influences on their quality. This method could also be useful for the determination of meat quality attributes as cooking loss or drip without evaporative losses.

Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells.

This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter range. A researcher familiar with the procedure can process up to 160 samples with four wells each within 3 d. The series described uses four plates with three concentrations per sample, which can be easily scaled to accommodate different sample sizes.

Factors influencing the 18O/16O-ratio in meat juices.

With respect to the question of whether the 18O/16O-ratio of meat water could be used for meat origin analysis, factors influencing its delta18O-value have been examined. The 18O/16O-ratio of meat water differs geographically, similar to known differences in precipitation and ground water. In Great Britain higher enrichments in 18O were found in southern samples and in Germany in northern samples. However, it was not possible to distinguish between British and German beef: their 18O/16O-ratios overlapped. British bovine samples the variation range of delta18O-values was 2.8 per thousand; in porcine samples it was 2.0 per thousand. British meat liquids from beef were enriched in 18O by 1.3 +/- 0.3 per thousand compared with pork. In porcine samples of litter mates with identical breeding and age at slaughter, the range in the delta18O-values among individuals was 2.4 per thousand. Experiments revealed significant influences of the meat's storage and handling conditions on the 18O/16O-ratio. After 10 h at 21.5 or 18.5 degrees C the delta18O-value increased by 0.4 or 0.3 per thousand per h, respectively, in samples (50 g) of chopped meat. The observed magnitude of changes might compensate for the geographical and seasonal differences. A precise origin assignment of the affected specimen on the base of delta18O-values of meat water is hence bound to be impossible.