|
What is CALUX® ?
Chemical Activated Luciferase gene eXpression
Dioxins (polychlorinated dibenzodioxins; PCDDs) are one of the most toxic man-made compounds known.
They are persistent environmental pollutants (POPs) and tend to accumulate in biota. Dioxin-like compounds such as polychorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) show similar properties. This group of chemicals, also known as polyhalogenated aromatic hydrocarbons (PHAHs), display a wide variety of toxic effects in mammals, birds, and fish. Among the toxic effects observed as a result of exposure to PHAHs are immunotoxicity, carcinogenicity, metabolic changes, and even death.
New EU-wide legislation implemented in July 2002, set limit values for dioxins and dioxin-like materials in feed and foodstuffs with the aim of reducing dioxins intake to European populations. Stakeholders at all levels of the food chains have an obligation to make sure that materials going into the food supply - from farm to fork - do not exceed these limits. Materials not meeting these limits are not allowed to be used or are classified as unfit for human consumption. Traditional analytical techniques for dioxins are too expensive and time consuming for food and feed screening. BioDetection Systems' Chemically Activated Luciferase Expression or CALUX® reporter gene bioassay offers a practical technology for monitoring compliance with the new limit values.
These highly toxic materials are very potent and believed to have significant toxicological effect at very low doses. The units of exposure are usually measured in picogrammes (pg) per kilogramme bodyweight. One picogramme or "pg" is one millionth of one millionth of a gramme or 0.0000000000001 grammes.
A human Tolerable Monthly Intake (TMI) of 70 pg/kg bodyweight/month was established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) at its fifty-seventh meeting (Rome, 5-14 June 2001) and which builds on the Tolerable Daily Intake (TDI) of 1-4 pg WHO-TEQ/kg body weight, established by the World Health Organisation (WHO) Consultation in 1998.
On 30 May 2001, the Scientific Committee on Food (SCF) of the European Union (EU) adopted an opinion on the Risk Assessment of dioxins and dioxin-like PCBs in food. The SCF established a group Tolerable Weekly Intake (TWI) for dioxins and dioxin-like PCBs of 14 pg Toxic Equivalent (WHO-TEQ) /kg bodyweight which is in line with the JECFA TMI described above.
For these recommended levels, the current dietary exposure to dioxins and dioxin-like PCBs exceeds the Tolerable Weekly Intake (TWI) or the Tolerable Daily Intake (TDI) for a considerable part of the European population.
To respond to this position, the EU has identified a "pressing need" for further action to avoid adverse health effects from dioxins and PCBs, including the reasons that bioaccumulation is continuing along the trophic chain and that the toxicological properties of dioxins and PCBs have previously been underestimated. In order to fulfil this need, the EU has developed a Community Strategy to reduce exposure to these materials and secure better protection of human health and the environment.
The objectives of the EU strategy are to assess the current state of the environment and the ecosystem including the food chains, to reduce human exposure to dioxins and PCBs in the short term, to maintain human exposure at safe levels in the medium to long terms and to reduce environmental effects from dioxins and PCBs.
A key cornerstone of this strategy will be the imminent setting of limit values for dioxins in feedstuffs and foodstuffs. In the initial phase, it currently appears as though the dioxin-like PCBs will not be included as it is thought that not enough is known about the extent of PCBs contamination in the food chains. However, this approach is inconsistent with the JECFA/WHO established tolerable intakes and the toxicological evidence available, both of which include the dioxin-like PCBs. The interim solution is to set limits based on the PCDDs and PCDFs (polychlorinated dibenzodioxins and dibenzofurans) only, with a view to including the dioxin-like PCBs no later than the end of 2004 and maybe substantially earlier.
Tables of the proposed limit values for the various feedstuffs and foodstuffs can be found at the EU's Euro-Lex website.
Monitoring compliance with feedstuffs and foodstuffs limit values will entail the undertaking of a large number of analyses.
|
SWITCH ON THE LIGHT!
Innovative DR CALUX® cells have been tailored so that they produce light in a dose responsive way when exposed to dioxins and dioxin-like chemicals. The switch that turns the light on is specific to compounds of groups of compounds that have the same action giving the DR CALUX® assay a selectivity and biological relevance that cannot be matched by instrumental techniques.
|
|
Mode of action
|
|
And now more technically...
The mechanism of action of PHAHs has been extensively studied over the past decades and DR CALUX® assay mimics this mechanism in a biologically relevant way.
PCDDs, PCDFs, and PCBs share a common mode of action. These compounds bind to an intracellular receptor, known as the aryl hydrocarbon (Ah) receptor. Binding to the Ah receptor is followed by transportation of the PHAH-Ah receptor complex into the nucleus of the cell and subsequent binding to specific sequences in the DNA.
These specific DNA sequences are called dioxin responsive elements (DREs). Binding of the PHAH-Ah receptor to the DRE triggers the expression of DRE associated genes. The toxicological impact of dioxins and related compounds starts with the observed change in gene expression. |
|
By using the knowledge obtained from studying the Mode of action of dioxin and dioxin-like compounds, we has designed a genetically modified cell-line (rat-hepatoma H4IIE) that contains the firefly luciferase gene as a reporter for the presence of PHAHs. The newly constructed cell-line still contains the complete machinery which is involved in the Mode of action of dioxins and dioxin-like chemicals. In addition, a plasmid (pGudLuc 1.1) has been incorporated in the cells containing mouse DREs coupled to firefly luciferase genes. |
|
 pGudLuc 1.1
|
|
|
|
The thus created stable cell-line expresses firefly luciferase genes in addition to genes that are normally expressed in the parent cells upon exposure to dioxins or dioxin-like compounds. As a consequence, these cells emit light which can very easily be quantified. The amount of light-production is related to the amount of dioxins or dioxin-like compounds in the exposure mixture. |
|
We named this new assay the
DR CALUX®
= Dioxin Responsive Chemical Activated Luciferase gene eXpression assay.
|
|
Using the DR CALUX® assay to detemine the amount of dioxin or dioxin-like compounds in a given matrix is rapid and straightforward.
After sample collection (1), a simple extraction method is used to extract the dioxins and dioxin-like chemical content (2). The extract is cleaned-up and fractionated if desired (3), after which the clean extract is dissolved in DMSO.
Meanwhile, DR CALUX® cells are cultured (4, 5) and finally grown in 96-well plates under standardised conditions.
Once a confluent monolayer is obtained, the cells are exposed to the diluted cleaned extracts (6-48 hrs) (6).
After lysation and adding luciferin, the luciferase activity is quantitated using a luminometer (7). |
|
 The procedure
|
|
Detected luminescense from the analysed samples is compared to the detected luminescense from a TCDD standard curve. After data handling (8), the amount of TCDD TEQs in the analysed sample is calculated and reported. |
|
|
|
|
|
|
DR CALUX® assay is the perfect screening tool for the detection of dioxin and
dioxin-like compounds in a wide variety of matrices. The advantages of the newly developed DR CALUX® bioassay as compared to traditional analysis of dioxins by HRGCMS are:
> Extremely sensitive
DR CALUX® assay is capable of detecting femtograms (10-15 grams or quadrillionths of a gram) of dioxin toxic equivalents (TEQs).
> Rapid
Using 96-well test systems allows the rapid analysis of large numbers of samples simultaneously.
> Easy sample clean-up / work-up
Compared to dioxin analysis using HRGCMS, extracted samples do not have to be as clean for this bioassay. Hence, sample clean-up and work-up is relatively straightforward.
> Small sample size
The extreme sensitivity of the DR CALUX® assay allows small sample sizes: only grams of sample are required.
> Much reduced cost compared to instrumental methods
Because DR CALUX® assay is straightforward to perform, uses small sample, gives rapid results and doesn’t need expensive apparatus, extensive dioxin analysis is cost effective.
> Applicable to a wide variety of matrices
DR CALUX® assay has succesfully been used to determine dioxin content in the following matrices:
» Food and Feed samples (Animal Feedstuffs, Plant and vegetable matter,
Animal fat, Oils, Milk, Egg, Cheese)
» Environmental samples (Soils / clays, Stack samples, Air samples, Sediments)
» Water samples (Surface water, Groundwater, Drinking water, Wastewater,
Process aqueous effluents, Sewage and sewage sludge)
» Tissue samples (Animal tissues, Human tissues, Blood)
» Other (Pure chemicals (pharmaceuticals, AgRochemicals, fine chemicals etc),
Research materials)
> Biologically relevant
Being cell based, DR CALUX® assay is more biologically relevant than instrumental techniques. The DR CALUX® assay operates through measuring the biological response elicited following binding at the AhR receptor in a living cell, the same receptor that is preserved in man, mammals, fish, birds etc. In the DR CALUX® cells, respiration and metabolisation are taking place as are the mechanisms of receptor binding. The net result is that the DR CALUX® assay, to a significant extent, mimics the biological process that is thought to be a significant part of the mechanism of dioxin toxicity. This mechanism of ligand-AhR binding, opens the way for using the DR CALUX® assay for pharmaceutical research. Materials which may be agonists or antagonists at the AhR receptor can be investigated in high rate screening.
> Analysis of total toxic equivalence of PHAH and PAH mixtures in samples
DR CALUX® assay is the perfect screening tool for assessing complex mixtures of dioxin-like chemicals in a wide variety of matrices. Instead of determining the number of individual congeners in a complex mixture, DR CALUX® assay analyses the total toxic equivalence of polyhalogenated aromatic hydrocarbon (PHAH) and polyaromatic hydrocarbon (PAH) mixtures in samples under investigation. Once a contaminated sample has been identified, congener specific identification of PHAHs and PAHs can be carried out using high resolution HRGCMS analysis. We are able to offer HRGCMS through its collaboration with RIKILT.
> Distinguish between stable and non-stable PHAHs or PAHs
DR CALUX® assay can be used to distinguish between stable and non-stable PHAHs or PAHs by variation of the time of exposure. The genetically modified H4IIE rat hepatoma cells are capable of metabolising non-stable PHAHs or PAHs since they contain the relevant biotransformation pathways. The metabolised PHAHs or PAHs do not bind to the Ah-receptor and therefore induction of luciferase will be absent. For example, exposing DR CALUX® cells for 6 hrs will result in expression of luciferase induced by both stable and non-stable PHAHs or PAHs. Exposure period of 24 hrs results in expression of luciferase induced by practically only stable compounds. In addition, adjusting the extraction and clean-up procedures is another tool to distinguish between different classes of PHAHs.
5. The Limit of Detection and Limit of Quantitation for the DR CALUX®,
Much can be hidden in the LODs and LOQs that some companies state. Often these are the LOD and LOQ of the core method when analysing standards – NOT true matrices. Some HRGCMS laboratories, for example, give results based on recoveries of spikes of less than 10%. In principle, anyone can obtain lower and lower LOD/LOQs by processing more and more matrix. However, this can introduce error and can result in the use of large quantaties of reagents and the generation of much waste. DR CALUX® bioassay is fundamentally extremely sensitive and to get the LOQs for almost all applications we only need to process a few grams of material. We states openly and transparently how much matrix must be processed to achieve an LOD/LOQ.
The DR CALUX® Bioassay (LOD and LOQ of 0.3 and 1.0 pM in each well respectivly)
>For determining LOD and LOQ you need to know that dimethylsulfoxide (DMSO) is normally used as the solvent vehicle in the CALUX® bioassay and all test samples including the different concentrations of reference materials and blanks are measured in triplicate on a 96-well microtiter plate. The standard deviation of the test sample triplicates in DMSO, are interpolated against the calibration curve of the reference material resulting in a concentration of the test sample versus a reference chemical. If the results of blank samples on many microtiter plates are combined, an overall standard deviation for the DMSO blanks (expressed as concentration of the reference material) can be determined. This is the “background noise” in the bioassay.
To statistically determine that a signal is a true signal above background, a safety factor is applied. This results in the Limit of Detection (LOD). The safety factor for the LOD that we use is 3x the standard deviation of DMSO blanks interpolated to the calibration curve of the reference chemical resulting in LOD in terms of a concentration of the reference chemical. This is done for a large number of microtiterplates which then takes account of small differences between different plates.
The same procedure is followed to derive a Limit of Quantitation except that a factor of 10x the standard deviation is used. Samples values above this 10x level are very highly unlikely to have a value the same as the DMSO blank.
This results for example in the DR CALUX® in a LOD and LOQ of 0.3 and 1.0 pM in each well respectively.
The extract (LOD and LOQ of 0.3 and 1.0 pg per extract respectively)
Although the LOD and LOQ of a specific matrix depends on the amount of material extracted, the LOD and LOQ of the extract in pg 2,3,7,8 TCDD can be determined for all matrices . With the core CALUX® LOD and LOQ given above, combined with the knowledge that a sample is diluted 125 times in the bioassay , a sample is normally dissolved in 25 µl DMSO and the molar mass of 2,3,7,8 TCDD is 322, the LOD and LOQ can readily be expressed in pg 2,3,7,8 TCDD TEQ for each test sample.
If a lower LOD and LOQ are required, we have an enhancement to the DR CALUX® bioassay which results in a LOD and LOQ of almost a factor three lower – this is without processing more matrix.
The normal procedure results for example in the DR CALUX® in a LOD and LOQ of 0.3 and 1.0 pg per extract respectively.
The samples (see table)
If you wish to know the LOD and LOQ that can be achieved for a particular sample you need to know how much matrix is used for the analysis. Whilst this may not be a big issue for animal feed, it is certainly an issue for human tissues where only limited amounts of material can be obtained. Lower and lower LODs and LOQs can be obtained if you process a lot of matrix, however this can result in unwieldy and impractical work-up procedures. We normally uses small practical quantities of matrix – typically 1 – 10 grams. Given the information above about the LOD and LOQ, matrix dependent LODs and LOQs can be determined (see table below for some typical examples)
|
matrix |
unit |
Normal amount for work-up |
pg LOD/matrix |
pg LOQ/matrix |
|
milk |
gram fat |
1 |
0.30 |
1 |
|
egg |
gram fat |
1.5 |
0.20 |
0.67 |
|
vegetable oil |
gram material |
3 |
0.10 |
0.33 |
|
fish oil |
gram fat |
1.5 |
0.20 |
0.67 |
|
cattle feed |
gram fat |
9 |
0.03 |
0.11 |
From the table it can be seen that the EU limit values for foodstuffs and feedstuffs can easily be achieved (a technique should be able to meaure half of the limit value). In the next few years, the limit values for feed and food in the EU may go down significantly. Constrained by reporting upper-bound values for individual congeners, already the vast majority of HRGCMS labs are over estimating the amount of dioxins in samples (actually reporting upper-bound levels which as sensitivity is not enough non-detects are being reported above the EU limit values). With lower limit values likely, HRGCMS may just not be sensitive enough for dioxins analysis in future.
If you want to know the specific LOD and LOQ for your matrix of interest please contact us. The LOD and LOQ achievable for samples delivered to us for small quantities may be insufficient for your needs as the amount of material becomes the limiting factor - not the analysis procedure. This happens for example with serum samples. Serum samples are normally delivered in quantities of 5 gram serum but because the TEQ is normally expressed as 2,3,7,8-TCDD TEQ per gram fat, the LOD and LOQ becomes dependent on the amount of fat present in the serum.
|
