Environmental chemistry towards food processing

TOXICOKINETICS: TOXICODYNAMICS

Absorption, Distribution, Metabolism, Excretion

For chemicals that are not genotoxic, the aim of hazard characterisation is generally to set a health-based guidance value. This is a level of exposure that is without appreciable risk to health over a defined period.

For plant protection products and veterinary medicines, the common health-based guidance value is the acceptable daily intake (ADI), which is an estimate of the amount of a chemical in food or drinking water, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable health risk to the consumer (Chronic Risk).

The health-based guidance values might be expressed as Acute Reference Dose (ARfD) when considering the risk deriving from chemicals with the potential of causing effects following short-term exposure. The ARfD relates to the amount of a substance in food or drinking water that can be ingested in a single day without appreciable health risk to the consumer (Acute Risk).

drinking water, expressed on a body weight basis, that can beingested in a period of 24h or less without appreciable health risk (Acute Risk).The health-based guidance value is calculated by identifying the NOAEL (no observed adverse effect level)and dividing it by the UNCERTAINTY FACTOR.The UNCERTAINTY FACTOR consider the variability of response to the risk between different species andwithin a single specie (intra-specie variability).

Exposure AssessmentAssessment of exposure to chemicals in food requires information on the occurrence of the chemical indifferent types of food, and on the amounts of those foods that are consumed by different population groups.To measure the occurrence of contaminants we have to measure residues:- Pre-regulation (drugs, residues etc.) information already- Post-regulation information: environmental contaminants and residues. It’s the chemicalconcentration that we have to measure in food present on the market.Consumption data have to be used in

Exposure assessments; they should cover the general population, as well as critical groups that are vulnerable or are expected to have exposures that are significantly different from those of the general population (e.g. infants, children, pregnant women or the elderly). The methodologies should take into consideration non-average individuals, such as those who consume large portions of specific food items or show loyalty to specific foods or brands of food containing the highest concentrations of the chemical of interest.

To carry out this kind of assessment, we need to know on one hand the consumption data of contaminants, and on the other hand, we need to have the consumption data. The critical groups non-average individuals, such as those who consume large portions of specific food items or show loyalty to specific foods or brands of food containing the highest concentrations of the chemical of interest.

Mixture of them may have interactions between them: the different components present in the mixture might have an addictive and synergistic interaction, for example if a particular contaminant is present in a complex mixture with other molecules, these molecules may amplify the toxigenicity effect of the contaminant so we will get a higher than expected effect. In the event that other molecules have an antagonistic effect on the contaminant, we have a lower than expected effect.

Bioassays are biological tests that examine the combined action of all natural and anthropogenic constituents of a particular sample on human health. So any test system that takes advantage of biological molecules to detect a particular analyte. To detect the effect on biological system we use cytosensors, based on the response of cultural living cells. These cells are exposed to a toxic molecule (or a molecule we're examining) and this may change the physiology of cell so that we can assess whether or not this.

1. Cell-based Bioassays
1. Cell viability and cytotoxicity assays

This kind of test measures whether toxic molecules can induce a loss of cellular structure or function. In this case we can use probes, which are molecules that we can somehow detect, and see if they're able to cross the lipid membrane or not. When we have a functioning lipid membrane, it is able to discriminate what enters and what does not; when we don't have a functional membrane that means that the membrane is damaged.

So if we exposed cells to an unknown molecule and we detect that the membrane is damaged, that means that the molecule we're examining is toxic for the cell because it's responding in a way that is detrimental to its structure and function.

A physiological way to evaluate cell viability and cell toxicity is by using dyes such as trypan blue or propidium iodide. These dyes are able to bind to DNA but cannot cross the cell membrane. If they enter the cell, it indicates that the membrane is damaged, allowing us to determine if the cell is viable or not.

Propidium iodide (PI) binds to DNA but does not cross the membrane. If the membrane is damaged, PI will stain the cell, as it only fluoresces when it binds to DNA.

Fluorescein diacetate (FDA) is a fluorescence molecule that, when processed inside the cell by the enzyme esterase, releases a fluorescent green light. Therefore, if the cell is viable, it will emit fluorescence.

By measuring the fluorescence, we can determine how many cells have been affected by the toxic molecule and how many are still living cells. We can also quantify the release of intracellular enzymes such as alkaline phosphatases or lactate dehydrogenases.

be inside the cell. If the membrane is damaged some enzymes may be released. The toxicity does not only generate the death of the cell but also induces other consequences, so we need to investigate other aspects to characterise the effect of the contaminant compounds.

2. Calcium signalling measurements

Cells receive a lot of stimuli from the external environment. On the cell membrane we have a lot of proteins, which have different functions, such as receptors that can be stimulated. When a particular molecule binds a receptor the signalling cascade starts.

One of this signal cascade that's important for us is the one related to calcium. Calcium is a mineral element which is stored in the endoplasmic reticulum (sarcoplasmic reticulum). The cells can translate the external signalling because calcium can be detected by proteins called calmodulins. The concentration of calcium in the cell is detected by these proteins; after this recognition between calcium and calmodulin we have

othersignalling cascade. Calcium is already present in the cell, we haveto measure the response of calcium (how it is released by the cell)when a toxic molecule is present. We can use fluorescentmolecules (Fluo-4); once they’re binding, the calcium can emitfluorescence.

The thing that we can do to understand if a particularmolecule is toxic or not for the cell is adding the fluorescent dye tothe culture (able to bind the calcium), adding the molecule wewant to examine and then we can measure thefluorescence emitted to understand if the molecule we are examiningis affecting the signal transduction. If the molecule affects the signal transduction that means that istoxic for our cells.

3. Reporter gene assays

We use this technique to understand if a particular molecule can affect a gene or not.This approached is based on reporter genes. Genes encode for proteins and enzymes and we canmeasure the activity of enzymes. We can measure colour for instance and some enzymes can emitlight.

Expose the cell to the molecule we want to examine and we measure the response of this reporter gene. If it codify for the enzyme luciferase (enzyme which is able to catalyse a bioluminescent reaction) we will get an emission of light. If our reporting system is responding to a particular molecule we're tasting, we get the emission of light. This process can be quantitive (amount of the light emitted is proportional to the concentration of the contaminant) and qualitative.

Transcriptomics fingerprinting technologies

The aim is to analyse the RNA sequences and to do so there're several approaches. PCR protocol DNA polymerase - DNA dependant there only able to read DNA sequences. To date, reverse-transcriptase polymerase chain reactions (RT-PCR) and DNA microarrays constitute the most popular transcriptomics techniques, but these traditional methods may soon be replaced by more advanced biotechnological methods involving