Evidence of in vitro metabolic interaction effects of a chlorfenvinphos, ethion and linuron mixture on human hepatic detoxification rates

Highlights

• Human detoxification of linuron and ethion is significantly delayed by their concomitance in a mixture including chlorfenvinphos.

• Cytochrome P450 enzymes mediate the human hepatic inhibitions observed.

• Dietary and environmental risk assessment processes should include in vitro metabolic interaction studies on relevant mixtures.

Abstract

General population exposure to pesticides mainly occurs via food and water consumption. However, their risk assessment for regulatory purposes does not currently consider the actual co-exposure to multiple substances. To address this concern, relevant experimental studies are needed to fill the lack of data concerning effects of mixture on human health. For the first time, the present work evaluated on human microsomes and liver cells the combined metabolic effects of, chlorfenvinphos, ethion and linuron, three pesticides usually found in vegetables of the European Union. Concentrations of these substances were measured during combined incubation experiments, thanks to a new analytical methodology previously developed. The collected data allowed for calculation and comparison of the intrinsic hepatic clearance of each pesticide from different combinations. Finally, the results showed clear inhibitory effects, depending on the association of the chemicals at stake. The major metabolic inhibitor observed was chlorfenvinphos. During co-incubation, it was able to decrease the intrinsic clearance of both linuron and ethion. These latter also showed a potential for metabolic inhibition mainly cytochrome P450-mediated in all cases. Here we demonstrated that human detoxification from a pesticide may be severely hampered in case of co-occurrence of other pesticides, as it is the case for drugs interactions, thus increasing the risk of adverse health effects. These results could contribute to improve the current challenging risk assessment of human and animal dietary to environmental chemical mixtures.

Introduction

Synthetic pesticides have helped to increase crop yields of modern agriculture for more than half a century. However, due to their widespread use as insecticides, herbicides, fungicides, fumigants and rodenticides, they are now considered as a major group of contaminants. For the general population, although pesticide use for elimination of pests is a significant route of indoor exposure (Van den Berg et al., 2012), dietary intake including water consumption is considered to be the main source of exposure to most pesticides (Cao et al., 2011, Damalas and Eleftherohorinos, 2011, Ding, 2014). Thus, food commodities may simultaneously contain different pesticide residues, resulting in an uninterrupted exposure of human populations to complex pesticide mixtures through their diet. Crepet et al. (2013) found that the French population is mainly exposed to 7 different pesticide mixtures composed of two to six compounds (among 79 targeted food pesticides). As the marketing authorization for a chemical substance is delivered at the European Union scale, it could be assumed that the whole European population is likely to be exposed to these same pesticide mixtures. Among these residues, a mixture including two organophosphorus compounds (chlorfenvinphos and ethion) banned since 2007 but not necessarily totally off the agricultural practice (Storck et al., 2017) and a substituted urea (linuron), was found to be frequently present in staple foods such as carrots and potatoes (see Fig. 1).

The organophosphorus insecticide chlorfenvinphos [2-chloro-1-(2,4-dichlorophenyl)vinyl diethyl phosphate] is a neurotoxic molecule which inhibits the acetylcholinesterase. This phospho-organic pesticide is transformed in mammals by a hepatic oxidative O-deethylation (Hutson and Wright, 1980). Moreover, chlorfenvinphos administration leads to microsomal enzyme induction and alterations of free amino acid concentrations in rat liver (Sedrowicz et al., 1996). In the same way, an in vivo study has revealed that chlorvenvinphos decreases the glutathione level and increases the concentrations of hydrogen peroxide and serum total glutathione in liver (Lukaszewicz-Hussain, 2011). Indeed, chlorfenvinphos liver metabolism is associated with cytochrome P450 (CYP) activities resulting in the generation of reactive oxygenated metabolites and oxidative stress (Swiercz et al., 2013). Esterase enzymes seem to play a secondary role in chlorfenvinphos metabolism (Ikeda et al., 1991).

Ethion (O,O,O′,O′-tetraethyl S,S′-methylene bis(phosphoro-dithioate), is also an organophosphorus insecticide, which presents the same mechanism of action, compared to chlorfenvinphos. Ethion is converted in the liver to its active oxygenated analog, ethion mono-oxon, via desulfuration thanks to cytochrome P-450 enzymes (Desouky et al., 2013). Further biotransformation of the product, through ester cleavage, is catalyzed by esterase enzymes (Nigg et al., 1993, Mahajna et al., 1996).

Linuron, [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] is a phenylurea herbicide widely used in agriculture. Human liver is suspected to be a target of linuron, as it induced DNA damages in rat liver (Scassellati-Sforzolini et al., 1997). Another study demonstrated that exposure to linuron leads to hepatocellular adenomas in rat (Santos et al., 2014). Interestingly, this compound was described to be activated in mammalian metabolizing cells leading to an increase of mutagenic properties (Federico et al., 2016). Finally, linuron was shown to be an aryl hydrocarbon receptor (Ahr) ligand and its agonistic activity leads to the induction of CYP1A genes’ expression (Takeuchi et al., 2008).

Risk assessment carried out across the world by authorities mainly focus on compounds belonging to the same chemical family, or possessing the same mechanisms of action (Reffstrup et al., 2010, Ragas and Holmstrup, 2013). In addition, assessment is only based on the evaluation of cumulative effects of these products, supposing the absence of potential effects concerning interactions between pesticides (European Food Safety Agency, 2012). Therefore, a wide thinking process has been started for more than 5 years to address the issue of risk assessment regarding the combined actions of substances on human health (Solecki et al., 2014, European Commission, 2014, Rider et al., 2013). An increasing number of experimental studies have been published in the last few years (Starr et al., 2012, Takakura et al., 2013, Carvalho et al., 2014, Orton et al., 2014, Cedergreen, 2014, Clarke et al., 2015, Spaggiari et al., 2016), helping to fill the gap in knowledge on this topic. More than a decade ago, Tang et al. (2002) already demonstrated a strong inhibition in the metabolism of carbaryl when it was simultaneously incubated with chlorpyrifos in human liver sub-cellular preparations. Similarly, Savary et al. (2014) showed that hepatic metabolic interactions occurred during the co-incubation of the pesticides endosulfan and methoxychlor. This phenomenon increases the residence time of the active compounds and thus their latent toxicity. While Savary studied these interactions effects through the activities of the CYP isoforms involved, Tang et al. (2002) used the same experimental strategy. However, they also demonstrated the occurrence of an interaction effect on the basis of calculated intrinsic clearance rates resulting from human liver microsome experiments. An alternative approach to evaluate a metabolic interaction between two compounds consists in comparing the intrinsic clearances of the product when incubated alone, or as a mixture using the substrate depletion approach (Donglu et al., 2007).

Human liver is the most important site of biotransformation in the body, primary culture of hepatocytes and hepatocyte subcellular preparations have proven to be suitable in vitro models for the investigation of the metabolism and metabolic interactions of environmental contaminants (Hodgson et al., 2014). Although liver microsomes support only a part of the whole metabolic process i.e. phase I metabolism, it continues to be the first-line model for metabolism study assays. Indeed, they are more readily available than hepatocytes and specifically adapted to CYP kinetic measurements. In order to highlight the part of phase II metabolism and cellular uptakes, primary culture of hepatocytes can be used as a complement as suggested by Houston and Carlile (1997), who demonstrated that both microsomes and hepatocytes might be suitable for the ranking of specific substrate hepatic intrinsic clearances in rats. Here, we investigated, for the first time, the effect of a co-incubation of multi-class pesticides present as a mixture in the French diet, especially on the human liver metabolism. In order to highlight the possible human metabolic interaction effects of the pesticide mixture, the analytical method previously developed by Kadar et al. (2017) will be applied to this in vitro study.