In Vitro Investigation on the Toxic Potentials of Commonly Used ...

APPLIED IN VITRO TOXICOLOGY Volume 1, Number 4, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/aivt.2015.0022

In Vitro Investigation on the Toxic Potentials of Commonly Used Synthetic Pyrethroids, Especially Esbiothrin ¨ zhan Ezgi Oztas, Bahar Ulus, and Gu¨l O

Abstract

Pyrethroids, including allethrin, bioallethrin, deltamethrin, and esbiothrin, are widely used to control a broad range of pests in agriculture, public health, and households as alternative to organophosphorus and organochlorine insecticides. Even if the pyrethroids are known to lead to asthma; allergic dermatitis; respiratory diseases; and immunosuppressive, reproductive, and carcionogenic effects with long-term exposure, data on their cancer risk are still conflicting. Moreover, exposure to pesticides including synthetic pyrethroids is highly possible both indirectly and directly through environmental contamination. Therefore, in the present study, it was aimed to assess in vitro toxic potentials of four synthetic pyrethroids. The half maximal inhibitory concentration (IC50) values of allethrin, bioallethrin, deltamethrin, and esbiothrin were 24.61, 18.26, 555.78, and 54.32 lM on human hepatocellular carcinoma cells (HepG2). By the Ames assay, they possessed weakly mutagenic potential with base-pair substitution. Also, they slightly induced DNA damage when assessed by the comet assay. This study was first to evaluate the genotoxic effects of esbiothrin on human liver cell. The results could be useful in determining their toxicological effects and taking precautions regarding their consumption. However, further studies should be needed to assess the possible risks to human health caused by pyrethroids. Key words: allethrin,

bioallethrin, deltamethrin, DNA damage, esbiothrin, hepatotoxicity

Introduction

S

ynthetic pyrethroids are extensively used to protect crops, livestock, and domestic animals against insects, weeds, and diseases due to their better photostability, enhanced insecticidal activity, and relatively low toxicity compared with natural pyrethrins, organochlorine, and organophosphorus insecticides.1,2 Potential health hazards for acute and chronic effects of synthetic pyrethroids have been reported in several studies. Their main toxic effects include asthma; allergic dermatitis; respiratory diseases; and immunosuppressive, reproductive, and carcionogenic (e.g., leukemia, multiple myeloma, and breast cancer) effects with long-term exposure.3–6 Although it was reported that the pyrethroids lead to the generation of free radicals, genotoxic and apoptotic effects resulting in gene mutation, chromosomal alteration, and DNA damage, data on cancer risk are still conflicting.6–8 The International Agency for Research on Cancer (IARC) classified synthetic pyrethroids as nonconclusive carcinogenic for humans (group 3), whereas the World Health Organization defined them as moderately dangerous insecticides.9,10 Allethrin, bioallethrin, deltamethrin, and esbiothrin are the most widely used synthetic pyrethroids (Table 1). Until today,

there are few data on their genotoxic effects. Especially, their hepatotoxic potentials are not available. Therefore, the present study aimed to assess the in vitro cyto- and genotoxic potentials of four synthetic pyrethroids in human hepatocellular carcinoma cell line (HepG2) and mutagenic potentials in Salmonella typhimurium. Materials and Methods Chemicals

Allethrin (Pestenal, 33396), bioallethrin (Pestenal, 31489), deltamethrin (Pestenal, 45423), and esbiothrin (Pestenal, 33309) standards (purity £99.2%) were obtained from SigmaAldrich. Stock solutions of pesticides were prepared by dissolving in dimethylsulfoxide (DMSO; 1%). All other chemicals in biochemical assays were obtained from Sigma-Aldrich and Merck in highest purity as available. Phosphate buffer solution (PBS; Ca-Mg free, pH 7.2), cell culture media, and all other supplements were purchased from Wisent Bioproducts, and sterile plastic materials were purchased from Greiner. Cell culture

HepG2 human hepatocarcinoma cell line (HB-8065) was purchased from American Type Culture Collection. The

Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey.

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IN VITRO TOXIC POTENTIALS OF PYRETHROIDS

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Table 1. Structures of Four Synthetic Pyrethroids9,10

as the concentration of sample caused 50% inhibition of enzyme activity of cells. For all concentrations, the protocol was performed in triplicates and every protocol was repeated at least twice to ensure reproducibility.

Structural formula

Genotoxicity (comet) assay

(RS)-3-allyl-2-methyl-4-oxocyclopent-2-enyl (1R)-cis,trans chrysanthemate Allethrin [1R, trans; 1R] + [1R, trans; 1S] + [1R, cis; 1R] + [1R, cis; 1S] (* ratio of 4:4:1:1) (CAS No.: 584-79-2) Bioallethrin [1R, trans; 1R] + [1R, trans; 1S] (* ratio of 1:1) (CAS No.: 584-79-2) Esbiothrin [1R, trans; 1R] + [1R, trans; 1S] (* ratio of 1:3) (CAS No.: 260359-57-5)

The assay principle is to measure DNA strand breaks in eukaryotic cells lysed with detergent and high salt, and embedded in agarose on a microscope slide. The cells were exposed to final concentrations of 10,300 lM for deltamethrin and 5–30 lM for allethrin, bioallethrin, and esbiothrin during 24 h. The exposure concentrations were equivalent to the maximum 50% cell death. Hydrogen peroxide (H2O2) (10 lM) and 1% DMSO were used as positive and negative controls, respectively. The number of DNA breaks was scored under a fluorescent microscope (Olympus BX53; Olympus) at 400· magnification using an automated image analysis system (Comet Assay IV; Perceptive Instruments). One hundred cells were scored per concentration and DNA damage to individual cells was expressed as a percentage of DNA in the comet tail (tail intensity %, TDNA%).12,13 Data were expressed as mean – standard error (SE) and analyzed by ANOVA using SPSS version 21.0 for Windows. The significance of differences between HepG2 cells untreated (control) and treated with the pyrethroids was calculated by Dunnett’s t-tests. P-value