Food safety: new approaches to analysis

Sure, it tastes good

von Prof. Dr Dr Alfonso Lampen, Dr Thorsten Buhrke, Hermann Broll

Food safety is a requirement stipulated by Section 14 of EU Basic Regu­lation (EC) No. 178/2004. Food safety monitoring activities conducted by state inspection agencies ensure nationwide compliance with the high levels of food safety in Germany. As well as monitoring microbial loads in foodstuffs, the agencies also focus on a wide range of chemical contaminants, such as dioxins. Whether the foodstuffs have been contaminated with these kinds of potentially toxic substances deliberately or inadvertently as a result of an accident is initially ­irrelevant to the agencies’ work: the job of the enforcement laboratories is to track down contami­nated food and make sure it is withdrawn from sale.

The number of chemical substances officially monitored as potential contaminants of foodstuffs is continuously increasing: as a result, food safety monitoring is a field that is facing a growing set of challenges. According to estimates, the European Union has approved a total of approximately 80,000 substances, which also have the potential to enter the human body via the food we eat. The presence of substances such as e.g. various pesticides is established using validated chemical analysis methods. These methods generally consume a lot of time, expenditure and resources. A further disadvantage is that a chemical analysis method of this kind will identify and/or quantify only a single, predefined analyte: such a method cannot establish the presence of related substances with comparable toxicological potential. Examples of such substances include the multifarious members of the PAH and PCB families or dioxins – which are so numerous that it is impossible to analyse every last one of them. Although potentially toxic, if a dedicated search is not made for these substances, conventional chemical analysis methods will not detect them. Ideally, therefore, one needs to develop innovative procedures that are not only faster, less costly and less resource-intensive to complete but which are also capable of detecting both individual substances as well as substance groups with similar toxicological properties – and thus permitting a ‘screening’ of the substance in question.

Enter ‘effect-directed analysis’ (EDA): in contrast to conventional chemical analysis, which investigates substances on the basis of their physical and chemical properties, EDA is based on the concept of detecting the effects of individual substances or groups of substances. The foundations of such an analysis are provided by existing biological systems, which trigger a biological signal when they interact with a substance contained in the foodstuff. The substance therefore achieves a biological effect via a kind of biosensor. One example of such a substance would be the aryl hydrocarbon receptor (AhR), a transcription factor that initiates the expression of multiple xenobiotic-metabolising enzymes by direct interaction with dioxin and dioxin derivatives. This occurs by the binding of the activated AhR to ‘dioxin responsive elements’ (DREs) – i.e. specific promoter sequences that are located above the AhR-dependent target gene. Genetic manipulation now offers the possibility of coupling the DRE with an entity known as a ‘reporter’: a typical reporter is the gene for a protein named ‘luciferase’, which is responsible for giving glow-worms their glow since it excites a substance named ‘luciferin’ to create light. If one now sets up a test system where the reporter gene is controlled by a DRE, then the concentration of dioxin or dioxin-like substances in a sample will correlate to the AhR-induced expression of luciferase. The quantity of luciferase protein formed is proportional to the conversion of luciferin and the associated emission of light, which is relatively simple to quantify with the aid of a luminometer (Figure 1; [1, 2]). Since only a few molecules are needed to activate the DRE, a test of this kind is highly sensitive and capable of detecting all AhR-activating substances in a specified extract for analysis.

Alongside the well-characterised AhR, other biological systems are also suitable for use as starting-points for designing reporter gene test systems. Members of the family of nuclear receptors, for example, are transcription factors that are activated by the binding of specific ligands. These natural systems are superbly qualified for detecting a wide range of substances or substance groups that are of interest for food safety monitoring. Similarly to AhR, for example, the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) also interact with a large number of foreign substances with an aromatic base structure – such as are contained in many pesticides, for example. Then again, other food contaminants – such as a number of mycotoxins (aflatoxin B1) or ergot alkaloids, for example – are also capable of activating CAR and/or PXR. The glucocorticoid receptor (GR) is also a suitable model for developing a reporter gene system: in this case, for the detection of a range of steroid hormones – such as might be deployed illegally within the meat production industry, for example. Finally, a test system based on the oestrogen receptor (ER?) could be used for detecting hormonally active substances. Receptor-induced effects present just one option for approaching solutions within effect-directed analysis. Other endpoints include cytotoxicity, for example, genotoxicity/mutagenicity, as well as reproductive toxicity.

Reporter gene test systems based on the above-mentioned nuclear receptors have existed for some time and have been utilised extensively within research for several years now. To make these systems capable of deployment in day-to-day food safety monitoring work, they must be developed further, – especially in terms of simplification and handling, and as regards the standardisation and automation of the test systems in question. One possible strategy for establishing standardised procedures is the principle of reverse transfection. This involves the prefabrication (“coating”) of microtitre plates with the plasmids needed for the respective test system – a process that can also be automated by using pipetting robots. This approach would not only permit the standardised production of larger batches of plates prepared in this way, but would also mean that these plates could be stored stably for prolonged periods without efficiency losses. For reverse transfection, the plates coated with plasmids need then only be populated with the cells and an appropriate transfection reagent, plus – as a second step – furnished with the extracts from the foodstuffs that are to be investigated. An analysis of the results is typically complete within 24 hours, depending on the test system deployed. At first, this period of time seems very long in contrast to conventional chemical analysis. Two points must be borne in mind, however: first, biological test systems can investigate many samples in parallel; second, the actual luminometer measurements taken at the end of the incubation period need mere minutes to complete.

The BfR is particularly interested in developing these kinds of effect-directed analytical procedures to a point where they are resilient and accurate enough to produce reliable test results in food safety monitoring. Ultimately, the goal is for biological test systems to supplement – not replace – conventional chemical analysis. The goal should be to establish biological procedures as screening methods that are applied as an initial step – such as when sample volume is high, for example, a scenario that makes the methods of instrumental chemical analysis impractical to apply because of time and/or cost considerations. Only those samples returning a positive result from biological screening would then need to have these results verified in a second step by applying conventional chemical analysis techniques (Figure 2). Overall, then, a combination of biological screening and chemical analysis would facilitate high sample throughput while keeping time and costs within justifiable limits. If we consider the steadily expanding remit and the growing challenges facing food safety monitoring, this is a goal well worthy of pursuing.

Literature
[1] Kuhn, K. et al. (2009) Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 26,1104-1112.
[2] Kuhn, K. et al. (2008) J. Food Prot. 71,993-999.

Foto: © istockphoto.com|Oliver Helbig