METAQUANT metagenomics platform. © INRA, BEAUCARDET William

Cocktail effects of toxic substances demonstrated in vitro

The cocktail effect of pesticides

For the first time, an experimental study has demonstrated a cocktail effect for a mixture of five pesticides found in human food. This means that these chemicals are more toxic when combined than alone. The results showed DNA damage on lines of human cells cultivated in vitro.

By Pascale Mollier, translated by Teri Jones-Villeneuve
Updated on 11/22/2016
Published on 08/31/2016

People are regularly exposed to low amounts of pesticide residue in food. According to a study by EFSA, nearly half of the samples tested in the EU in 2008 contained pesticides. Often, several pesticides were detected: 27% of the samples contained at least two, while 9% contained more than four. Concerns about a possible synergistic effect between pesticides are not new, but until now, there was no experimental proof relevant to humans. This is no longer the case thanks to the genotoxicity test (1) developed by INRA’s ToxAlim Joint Research Unit in Toulouse.

Synergistic effect shown in tests on pesticide cocktails

A synergistic effect was demonstrated based on a combination of five pesticides (2) mainly found in fruit and vegetables. When examined alone, only two of the five chemicals have genotoxic effects (3) on liver cell lines after 24 hours of exposure. The more toxic of the two, fludioxonil, starts to affect cells from a concentration of 4 micromolar. An equal micromolar combination of all five chemicals is genotoxic from 3 micromolar, which amounts to 0.6 micromolar of each chemical. This reflects a clear synergistic effect because none of the five chemicals is toxic alone at a concentration of 0.6 micromolar. When the combination of actual concentrations found in food (4) are tested, toxic effects appear from 30 micromolar concentrations. As with the study of equal concentrations, the effect observed at 30 micromolar was higher than the effect for the chemicals alone.

Seven pesticide cocktails are most common in food

The seven pesticide combinations tested in this study are those we are most frequently exposed to through our food. They contain 25 chemicals found in various pesticides, of the 300 currently used in agriculture. They were identified through a prior study (5) which established two types of statistical data: consumption data (6) indicating the overall diet of French consumers and data regarding the pesticide contamination of food (7). One of these cocktails showed genotoxicity on just one of the four cell lines tested (liver, kidney, colon, nervous tissue).

Better identification of chemicals to ban

“Our results on cell lines demonstrate a genotoxic effect of fludioxonil and cyprodinil on human liver cell lines, as well as a synergistic effect between the two chemicals,” says Marc Audebert. “However, the results cannot be extrapolated in vivo. The amounts to which we are exposed are considered to be well below levels that would be toxic to a person, although exact figures are difficult to establish.

“First and foremost, our work focused on methods used to analyse potentially toxic pesticides. This analysis should be regularly updated as new toxicity tests are developed. Furthermore, those chemicals revealing toxicity and a resulting cocktail effect are the first that should be banned in any pesticide use reduction plan. Six of the 25 substances tested in the cocktails have actually been banned since the start of the study.

“We have effectively shown that the cocktail effect is real, increasing interest in understanding how the phenomenon works. We believe that certain substances may change how others are metabolised, thereby speeding up formation of genotoxic intermediaries.”

(1) This test makes it possible to quantify breaks in DNA via a biomarker: a histone that is phosphorylated when the DNA is damaged (see the study introduction page).

(2) The cocktail is composed of four fungicides – cyprodinil, fludioxonil, procymidone and iprodione – and one insecticide, lambda-cyhalothrin.

(3) Genotoxic effect: DNA damage.

(4) 42% procymidone, 33% iprodione, 16% cyprodinil, 9% fludioxonil and 1% lambda-cyhalothrin.

(5) PERICLES programme, Anses, INRA’s ToxAlim and Met@risk Joint Research Units

(6) 2006 INCA 2 survey of 4000 respondents.

(7) Data from DGCCRF surveillance plans.

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Animal Health
Associated Centre(s):
Occitanie-Toulouse

Reference

Graillot V., Takakura N., Le Hegarat, L., Fessard V., Audebert M. and Cravedi J-P. 2012. Genotoxicity of pesticide mixtures present in the diet of the French population. Environmental and Molecular Mutagenesis 53:173-184.

Test de génotoxicité sur cellules réalisé en plaque de 96 puits. Les cassures dans l'ADN occasionnées par le produit toxique testé sont mises en évidence par la phosphorylation concomitante d'une histone particulière. En rouge, l'ADN ; en vert, l'histone phosphorylée.. © INRA, Marc Audebert

a new genotoxicity test

The genotoxicity test, developed by Marc Audebert, consists in identifying double-strand DNA breaks in cells. Here, one of the histones (1) is phosphorylated. The test enables scientists to detect this phosphorylation through antibodies. It was developed using 96-well plates so that multiple combinations of substances at various concentrations can be tested quickly. The DNA damage is characteristic of precancerous cells, and the biomarker used in the test is being increasingly used in the field of oncology.

(1) Histones are proteins that associate with DNA and are involved in gene expression.