Elsevier

Science of The Total Environment

Volume 647, 10 January 2019, Pages 1052-1057
Science of The Total Environment

Interlaboratory evaluation of the genotoxic properties of pencycuron, a commonly used phenylurea fungicide

https://doi.org/10.1016/j.scitotenv.2018.08.067Get rights and content

Highlights

  • Pencycuron genotoxicity was investigated in collaboration of two laboratories.

  • The fungicide was able to induce statistically significant DNA damage.

  • Inter-lab variability was observed in comet assay results.

  • Further studies on the genotoxic characteristics of pencycuron are required.

Abstract

Pencycuron, a phenylurea-type antifungal agent, is used in agriculture worldwide for inhibiting the growth of various fungal pathogens of crops. Pencycuron residues were found in vegetables, soil and drinking water. Accordingly, both occupational and consumer exposure can be expected and may be significant. However, human toxicity studies on its genotoxic, mutagenic or carcinogenic potential are lacking. Therefore, a collaborative study was performed in two laboratories to investigate whether pencycuron exposure can induce DNA damage.

The genotoxic effect of 0–100 μg/ml pencycuron in in vitro cultures of human mononuclear white blood cells (MWBCs) and human hepatocytes (HepG2) was detected by cytokinesis-block micronucleus assay and comet assay.

The combined results of the two labs showed a dose-dependent DNA damage detected by micronucleus frequency, which reached statistical significance at 100 μg/ml concentration after 21-h exposure in HepG2 cells (p = 0.048). Significant genotoxic effect could also be observed in the comet assay from 50 μg/ml concentration in MWBCs, and at 100 μg/ml concentration in HepG2 cells in one lab. Nevertheless, this finding was not confirmed by the other lab in HepG2 cells, where Fpg-dependent oxidative DNA damage could also not be detected.

The results indicate that pencycuron may have DNA-damaging potential as well as point out inter-laboratory variability that calls for further studies to confirm the genotoxicity of this fungicide.

Introduction

Agrochemicals, developed to preserve the health of plants, may pose potential health risks to human beings. Chronic pesticide exposure is of great concern, especially among agricultural workers, supported by the growing number of reports linking pesticides to a wide variety of diseases, from cancer to reproductive and developmental disorders. In developing countries, it has been estimated that tens of millions of agricultural workers were directly exposed to pesticides through spraying, mixing, handling and preparing (Goldman, 2002). Furthermore, a large proportion of them have neither information nor training about the safe use of pesticides. There is also a global concern about the exposure of the general population to low levels of pesticide residues through food and water, as well as through their presence in the environment as persistent organic pollutants (POPs) (Aktar et al., 2009). Novel pesticides with increased efficacy and reduced environmental impacts have continued to be developed, but new products are frequently released to the market without sufficient data on their health hazards.

Pencycuron is a phenylurea fungicide developed by the Bayer Crop Science (Leverkusen, Germany) in 1976 and marketed under the brand name Monceren (European Chemicals Agency, 2014; Krämer et al., 2012). It is used in agriculture worldwide for inhibiting the growth of various fungal pathogens such as Rhizoctonia solani that invades potato, rice and cotton, and Pellicularia spp. that infect lettuce and tomato crops (Buysens et al., 2015; Tomin, 2009). It can be applied as a foliar spray, dust, or incorporated into the soil. Pencycuron is moderately persistent in soil and water systems. In Europe, it has been found at concentrations of 0.01 mg/kg in potato and lettuce, 0.02 mg/kg in soils, and 0.005 mg/kg in drinking water. Accordingly, both human occupational and consumer exposure can be expected to be notable. The European Food Safety Authority (EFSA) set the maximum residue limit for pencycuron in potato as 0.1 mg/kg and the acceptable daily intake (ADI) as 0.2 mg/kg bw per day, respectively (EFSA, 2010).

Pencycuron has low acute toxicity in rats and mice. It has been reported to have neither mutagenic nor carcinogenic effect in bacterial and animal models (CLH Report, 2014); however, a recent study has shown that a pencycuron containing fungicide formulation (Monceren 250 SC) exerts genotoxic effects in zebra fish (Danio rerio) during early stages of development (Ku-Centurion et al., 2016). Apart from this finding, human data on its genotoxic, mutagenic or carcinogenic potential are lacking.

Genotoxicity tests provide important information on risk for cancer and other degenerative diseases. There are several biomarkers which can help to assess the genotoxic properties of xenobiotics, among them the formation of DNA strand breaks and micronuclei (MN). Primary DNA damage indicated by DNA strand breaks in individual cells can be sensitively evaluated by the comet assay (Pu et al., 2015), Micronuclei, that can be formed from acentric chromosomal fragments or whole chromosomes left behind during mitotic cellular division, can be detected by the cytokinesis-block micronucleus (CBMN) assay which is a standardized, sensitive and simple laboratory technique to evaluate genomic damage in isolated cells (Nersesyan et al., 2016). Numerous human biomonitoring studies detected an increase in DNA strand breaks and MN frequency as a result of exposure to pesticides (Bolognesi and Holland, 2016; Intranuovo et al., 2018; Valverde and Rojas, 2009; Yang et al., 2014).

The aim of the present study was to investigate whether pencycuron exposure can lead to increased DNA damage in in vitro human cell models evaluated by both the cytokinesis-block micronucleus (CBMN) assay and the comet assay.

Section snippets

Experimental design

The experiments were carried out in the collaboration of the University of Debrecen, Debrecen, Hungary and the Ospedale Policlinico San Martino, Genoa, Italy, fostered by the Diagnosis, Monitoring and Prevention of Exposure-Related Noncommunicable Diseases (DiMoPEx) COST Action.

Chemicals

Pencycuron and chemicals used for the assays were purchased from Sigma-Aldrich Chemie GmbH (Heidelberg, Germany), the cell culture medium and the supplements were obtained from Gibco (Paisley, UK). The acetomethoxy

Pencycuron-induced cytotoxicity

The viability of cells treated with pencycuron for 1 h or used as controls was found to induce minimal decreases in cell viability over the tested concentration range. Both cell types showed high initial viability (>93%). 1-h exposure to pencycuron proved to induce minimal cell death in a concentration dependent manner (Fig. 1). Statistically significant decrease of the cell viability could not be observed in either cell types, which indicates negligible cytotoxic effect of pencycuron in the

Discussion

Pencycuron is a phenylurea-type fungicide commonly used for controlling fungal pathogens of crops in agriculture, but its genotoxic and mutagenic activity in human cells is poorly investigated. In the present interlaboratory study, our aim was to examine the possible DNA-damaging effect of pencycuron in human mononuclear white blood cells and cultured human HepG2 cells utilizing genotoxicity methods such as cytokinesis-block micronucleus assay and alkaline comet assay and to describe the

Acknowledgements

This collaborative work was carried out with the support of the Diagnosis, Monitoring and Prevention of Exposure-Related Noncommunicable Diseases (DiMoPEx) COST Action project (CA15129). The authors would like to thank Paola Roggieri and Andrea from Sciutto for his precious assistance in laboratory work.

Conflict of interest

The authors declare no conflicts of interest.

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    However, several negative aspects are associated with this agricultural practice such as the emergence of resistant species of fungi and toxic effects to non-target organisms (Al-balushi et al., 2018; Fisher et al., 2018). Indeed, various types of toxicity related to exposure to relatively high pesticide concentrations have been noticed such as neurotoxicity in zebrafish (Cao et al., 2019), embryotoxicity and teratogenicity in Xenopus (Wu et al., 2018), hepatotoxicity (Schmidt et al., 2018), spermatotoxicity (Pant and Srivastava, 2003), nephrotoxicity (Georgiadis et al., 2018) and hematotoxicity (Hashem et al., 2017) in rats, and in vitro cytotoxicity and genotoxicity in human white blood cells (Nagy et al., 2019). Besides the impact on crop yield, fungi are also responsible for human and animal diseases.

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The first two authors contributed equally to this work.

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