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METAQUANT metagenomics platform. © INRA, BEAUCARDET William

Cocktail effects of toxic substances demonstrated in vitro

Pesticide cocktail effect confirmed on a human nuclear receptor

Seven pesticide cocktails found in French foods show an effect on a nuclear receptor involved in the body’s detoxification mechanisms. For certain cocktails, the effect is stronger when the substances are combined than when separate. These results, confirmed during in vivo tests, have significant implications with regards to risk assessment procedures for chemical products.

By Pascale Mollier, translated by Teri Jones-Villeneuve
Updated on 11/22/2016
Published on 08/26/2016

In addition to the abovementioned study (1), the seven cocktails to which French consumers are most exposed (2) were analysed using another biological test to examine their binding to a major physiological nuclear receptor, PXR (3), in human liver cells. This receptor recognises certain exogenous (xenobiotic) substances that bind to it. This binding activates detoxification pathways and sets off programmed cell death or survival. The extra-physiological activation of this receptor, which also acts as a receptor for numerous hormones, can lead to metabolic and endocrine disorders and even carcinogenic risks.

Supra-additive effects observed for one cocktail

For one of the seven cocktails tested (4), the combined effect on the PXR is stronger than when the five substances are found alone. Remarkably, it was the same cocktail as that which had supra-additive effects in the genotoxicity test (1).

This supra-additive effect is observed when the five substances are found in equal proportions. However, when the substances are examined together in the proportions actually found in food (5), the combination becomes infra-additive, which means that it is less active with regards to activating the PXR receptor than each separate substance.

This complex effect underlines the importance of studying the effects of combinations of substances in the proportions found in the real world.

Another cocktail shown to have infra-additive effects

A cocktail of five fungicides and an organophosphate pesticide (6) showed infra-additive effects in PXR binding.  This effect was generally observed for both equimolecular concentrations and for exposure levels.

For the other cocktails, the effect of combinations was equal to the sum of the substances’ effects, indicating that there are neither positive nor negative interactions between the substances. It should be noted, however, that all the cocktails affect the PXR and are therefore potential sources of xenohormone-related disorders in the body.

“These results demonstrate the reality and complexity of cocktail effects between pesticides (and other chemical products) in different biological tests and confirm the need to continue studying the toxicity of xenobiotic combinations, especially those in food. These toxicological interactions could have unforeseeable harmful impacts on health as current regulations are based solely on individual products,” says Roger Rahmani, who coordinated this research.

(1) See Article 1 of this report.

(2) Cocktails identified in the PERICLES Programme (ANSES, INRA’s TOXALIM and Met@risk units), using consumer data indicating typical French diets (Inca2 survey in 2006 of 4000 individuals) and data on pesticide contamination of food (data obtained from DGCCRF monitoring).

(3) PXR: Pregnane X receptor.

(4) Combination observed especially in fruit and vegetables, composed of four fungicides (cyprodinil, fludioxonil, procymidone, iprodione) and one insecticide (lambda-cyhalothrin).

(5) 42% procymidone, 33% iprodione, 16% cyprodinil, 9% fludioxonil and 1% lambda-cyhalothrin.

(6) Combination found in table grapes, composed of Fenhexamid 52%, Pyrimethanil 31%, Fenitrothion 9%, Triadimenol 6%, Quinoxyfen 2%, Penconazole 1%.

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Animal Health
Associated Centre(s):
Provence-Alpes-Côte d'Azur, Occitanie-Toulouse

Reference

Georges de Sousa, Ahmad Nawaz, Jean-Pierre Cravedi and Roger Rahmani. 2014. A Concentration Addition Model to assess activation of the Pregnane X Receptor (PXR) by pesticide mixtures found in the French diet. Toxicological Sciences, 141(1), 234–243.

Structure du complexe entre le récepteur PXR et deux de ses ligands à effets supra-additifs : un oestrogène (en bleu) et un pesticide (en orange). Les pointillés en rouge figurent les interactions de stabilisation mutuelle entre les deux ligands. © Nature Communications

A molecular explanation for supra-additive effects

A study by the French National Institute of Health and Medical Research (Inserm), to which Roger Rahmani’s team contributed, used crystallography to gain insight on the structure of the assembly created by the nuclear receptor PXR and its ligands. The study revealed that certain ligands, especially oestrogens and pesticides, mutually stabilise themselves in the receptor-binding pocket. The resulting “supramolecular” ligand shows increased affinity for the receptor via positive cooperativity. These results offer a molecular-level explanation of how a complex combination of certain substances can exacerbate their effects.

This original research was published in 2015 in Nature Communications.

Delfosse V. et al. 2015. Synergistic activation of human pregnane X receptor by binary cocktails of pharmaceutical and environmental compounds. Nature Communications. 2015 Sep 3;6:8089. DOI: 10.1038/ncomms9089

Expanding the field of study

The European EuroMix project, launched as part of the Horizon 2020 programme, has allowed scientists to expand their study of chemical cocktails to a large range of compounds, including not just pesticides but PCB, dioxin, Bisphenol A and more that are found in European food or the environment. Because we are exposed to a nearly infinite number of combinations over the course of our lifetimes, the study will include a phase to identify key combinations.

These combinations will then be experimentally tested and the results used to evaluate the health impacts of overall and cumulative exposure. A battery of biological tests (hepatic, immune and developmental effects, etc.) adapted to analysing chemical combinations will be instituted, with the most suitable tests validated by comparing them against current tests used on lab animals. The Dutch National Institute for Public Health and the Environment (RIVM) is the project coordinator while INRA’s TOXALIM unit is the beneficiary.