From in vitro hepatic metabolic studies towards human health risk assessment: Two case studies of diuron and carbosulfan

Risk assessment of environmental pollutants is an absolutely essential tool in the overall process of protecting public health. Risk assessment needs reliable scientific information and one source of information is the characterization of metabolic fate and toxicokinetics of environmental pollutants. The aim of in vitro characterization is to produce relevant information on metabolism and interactions to anticipate and ultimately predict what happens in vivo in humans. Because human data is most appropriate to improve human risk assessment, the best option is to rely upon human-derived in vitro models and obtain quantitative toxicokinetics data from in vitro systems for the comparison between species or individuals. In short, based on our studies of pesticide metabolism and interactions, we have used in vitro metabolism data in human and animal hepatic in vitro models and clearance testing data to calculate chemical-specific adjustment factors, instead of fixed uncertainty factors, to be employed as an alternative and more realistic model for human health risk assessment of pesticides and other environmental pollutants.     

Researches on the evaluation of pesticide safety in humans using a pharmacokinetic approach

Similar to the pharmaceutical compounds, pesticides require human safety assessment for their registration and distribution; however, it is absolutely impossible to assess human safety by dosing humans with pesticides. Thus, how to appropriately evaluate the safety of pesticides in humans remains a great subject of debate. In this article, we present some examples of pesticide toxicity studies that identify species differences in toxicity and evaluate human safety by applying combinations of novel in vivo, in vitro, and in silico techniques to separately assess the key toxicodynamic (i.e., sensitivity) and/or toxicokinetic (i.e., exposure) factors. Because it is scientifically sound, the safety assessment strategy illustrated for three compounds in this article is expected to play an important role in the human safety assessment of agricultural compounds.     

Selectivity of diacylhydrazine insecticides to the predatory bug Orius laevigatus: in vivo and modelling/docking experiments

BACKGROUND: Knowledge of pesticide selectivity to natural enemies is necessary for a successful implementation of biological and chemical control methods in integrated pest management (IPM) programmes. Diacylhydrazine (DAH)‐based ecdysone agonists, also known as moulting‐accelerating compounds (MACs), are considered to be a selective group of insecticides, and their compatibility with predatory Heteroptera, which are used as biological control agents, is known. However, their molecular mode of action has not been explored in beneficial insects such as Orius laevigatus (Fieber) (Hemiptera: Anthocoridae). RESULTS: In this project, in vivo toxicity assays demonstrated that the DAH‐based RH‐5849, tebufenozide and methoxyfenozide have no toxic effect against O. laevigatus. The ligand‐binding domain (LBD) of the ecdysone receptor (EcR) of O. laevigatus was sequenced, and a homology protein model was constructed that confirmed a cavity structure with 12 α‐helices, harbouring the natural insect moulting hormone 20‐hydroxyecdysone. However, docking studies showed that a steric clash occurred for the DAH‐based insecticides owing to a restricted extent of the ligand‐binding cavity of the EcR of O. laevigatus. CONCLUSIONS: The insect toxicity assays demonstrated that MACs are selective for O. laevigatus. The modelling/docking experiments are indications that these pesticides do not bind with the LBD‐EcR of O. laevigatus and support the supposition that they show no biological effects in the predatory bug. These data help in explaining the compatible use of MACs together with predatory bugs in IPM programmes. Copyright © 2012 Society of Chemical Industry     

Predicting mammalian metabolism and toxicity of pesticides in silico

Pesticides must be effective to be commercially viable but they must also be reasonably safe for those who manufacture them, apply them, or consume the food they are used to produce. Animal testing is key to ensuring safety, but it comes late in the agrochemical development process, is expensive, and requires relatively large amounts of material. Surrogate assays used as in vitro models require less material and shift identification of potential mammalian toxicity back to earlier stages in development. Modern in silico methods are cost‐effective complements to such in vitro models that make it possible to predict mammalian metabolism, toxicity and exposure for a pesticide, crop residue or other metabolite before it has been synthesized. Their broader use could substantially reduce the amount of time and effort wasted in pesticide development. This contribution reviews the kind of in silico models that are currently available for vetting ideas about what to synthesize and how to focus development efforts; the limitations of those models; and the practical considerations that have slowed development in the area. Detailed discussions are provided of how bacterial mutagenicity, human cytochrome P450 (CYP) metabolism, and bioavailability in humans and rats can be predicted. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Fipronil metabolism,oxidative sulfone formation and toxicity among organophosphate‐ and carbamate‐resistant and susceptible western corn rootworm populations

Fipronil toxicity and metabolism were studied in two insecticide‐resistant, and one susceptible western corn rootworm (Diabrotica virgifera virgifera, LeConte) populations. Toxicity was evaluated by exposure to surface residues and by topical application. Surface residue bioassays indicated no differences in fipronil susceptibility among the three populations. Topical bioassays were used to study the relative toxicity of fipronil, fipronil + the mono‐oxygenase inhibitor piperonyl butoxide, and fipronil's oxidative sulfone metabolite in two populations (one resistant with elevated mono‐oxygenase activity). Fipronil and fipronil‐sulfone exhibited similar toxicity and application of piperonyl butoxide prior to fipronil resulted in marginal effects on toxicity. Metabolism of [¹⁴C]fipronil was evaluated in vivo and in vitro in the three rootworm populations. In vivo studies indicated the dominant pathway in all populations to be formation of the oxidative sulfone metabolite. Much lower quantities of polar metabolites were also identified. In vitro studies were performed using sub‐cellular protein fractions (microsomal and cytosolic), and glutathione‐agarose purified glutathione‐S‐transferase. Oxidative sulfone formation occurred almost exclusively in in vitro microsomal reactions and was increased in the resistant populations. Highly polar metabolites were formed exclusively in in vitro cytosolic reactions. In vitro reactions performed with purified, cytosolic glutathione‐S‐transferase (MW = 27 kDa) did not result in sulfone formation, although three additional polar metabolites not initially detectable in crude cytosolic reactions were detected. Metabolism results indicate both cytochromes P450 and glutathione‐S‐transferases are important to fipronil metabolism in the western corn rootworm and that toxic sulfone formation by P450 does not affect net toxicity. © 2000 Society of Chemical Industry     

Framework for defining pesticide maximum residue levels in feed: applications to cattle and sheep

BACKGROUND

Pesticide residues in animal feed can endanger animal health and compromise the safety of livestock products for human consumption. Even though policymakers such as the European Union and the World Health Organization have established maximum residue levels (MRLs) for pesticides in both human food and animal feed, there is no systematic management of pesticides in animal feed that considers the entire supply chain. In response, we propose a framework for defining consistent MRLs for pesticides in animal feed that assesses the impact of defined MRLs on upstream (e.g., MRLs in feed crops) and downstream (e.g., MRLs in livestock products) sectors of the livestock-product supply chain.

RESULTS

The MRLs determined for the selected pesticides in the feed of cattle and sheep as case study animals indicate that lipophilic pesticides tend to have lower MRLs than hydrophilic pesticides, primarily due to the relatively high toxicity and biotransfer factors of lipophilic pesticides. In addition, we observe that, primarily for lipophilic pesticides, upstream and downstream regulations are not aligned in terms of defining MRLs in feed using current MRLs in crops with relevance to feed and foods of animal origin.

CONCLUSION

Some of the current pesticide regulations in the livestock-product supply chain need to be re-evaluated to ensure that MRLs in the upstream sector (i.e., crops) do not result in unacceptable residues in the downstream sector (i.e., MRLs in livestock products affecting animal and human health). Finally, we provide recommendations for optimizing the derivation of MRLs in feed, including the evaluation of residue fate during feed and food manufacturing processes. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Recent progress of highly efficient in vivo biological screening for novel agrochemicals in China

This paper describes the recent progress of in vivo biological screening for pesticides in China. According to the criteria, including the severity of damage caused by pests and the economic value of the crops, the investigated insects, pathogens, herbs and other species in the agricultural field were selected as the main screening targets for pesticides. Corresponding in vivo microscreening methods have been established and applied in the pesticide screening procedure, which has higher reproducibility, a shorter time and greater efficiency that offset the drawbacks of conventional methods for pesticide screening. Copyright © 2009 Society of Chemical Industry     

Current- and past-use pesticide prevalence in drainage ditches in the Lower Mississippi Alluvial Valley

BACKGROUND: Pesticide application is common in agriculture and often results in applied pesticides entering adjacent aquatic systems. This study seasonally analyzed a suite of 17 current‐ and past‐use pesticides in both drainage waters and sediments to evaluate the prevalence of pesticides in drainage ditches across the Lower Mississippi Alluvial Valley (LMAV). RESULTS: There were significantly higher concentrations (P < 0.05) of current‐use than past‐use pesticides; however, there were consistently high numbers of detections of past‐use pesticides in sediments. Sediment pesticide concentrations were an order of magnitude higher (150–1035 µg kg^?1) than water samples (6–20.9 µg L^?1). Overall, 87% of all samples analyzed for current‐ and past‐use pesticides were non‐detects. p,p′‐DDT was detected in 47.5% of all drainage waters and sediments sampled. There were significant correlations (0.372 ≥ r²≤0.935) between detected current‐use water and sediment concentrations, but no significant correlations between past‐use water and sediment concentrations. CONCLUSION: Overall, there was a high percentage (87%) of sediment and water samples that did not contain detectable concentrations above the lower limit of analytical detection for each respective pesticide. This lack of pesticide prevalence highlights the improved conditions in aquatic systems adjacent to agriculture and a potential decrease in toxicity associated with pesticides in agricultural landscapes in the LMAV. Copyright © 2011 Society of Chemical Industry     

Assessment of species specificity of moulting accelerating compounds in Lepidoptera: comparison of activity between Bombyx mori and Spodoptera littoralis by in vitro reporter and in vivo toxicity assays

BACKGROUND: Dibenzoylhydrazine analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. A notable feature is their high activity against lepidopteran insects, raising the question as to whether species‐specific analogues can be isolated. In this study, the specificity of ecdysone agonists was addressed through a comparative analysis in two important lepidopterans, the silkworm Bombyx mori L. and the cotton leafworm Spodoptera littoralis (Boisd.). RESULTS: When collections of non‐steroidal ecdysone agonists containing different mother structures (dibenzoylhydrazine, acylaminoketone, tetrahydroquinoline) were tested, in vitro reporter assays showed minor differences using cell lines derived from both species. However, when compounds with high ecdysone agonist activity were examined in toxicity assays, larvicidal activity differed considerably. Of note was the identification of three dibenzoylhydrazine analogues with > 100‐fold higher activity against Bombyx than against Spodoptera larvae. CONCLUSION: The present study demonstrated that species‐specific ecdysone‐agonist‐based insecticides can be developed, but their species specificity is not based on differences in the activation of the ecdysone receptor but rather on unidentified in vivo parameters such as permeability of the cuticle, uptake/excretion by the gut or metabolic detoxification. Copyright © 2010 Society of Chemical Industry     

Eco‐efficiency as a strategy for optimizing the sustainability of pest management

Agricultural industrialization and the subsequent reliance on pesticides has resulted in numerous unintended consequences, such as impacts upon the environment and by extension human health. Eco‐efficiency is a strategy for sustainably increasing production, while simultaneously decreasing these externalities on ecological systems. Eco‐efficiency is defined as the ratio of production to environmental impacts. It has been widely adopted to improve chemical production, but we investigate the challenges of applying eco‐efficiency to pesticide use. Eco‐efficiency strategies include technological innovation, investment in research and development, improvement of business processes, and accounting for market forces. These components are often part of integrated pest management (IPM) systems that include alternatives to pesticides, but its implementation is often thwarted by commercial realities and technical challenges. We propose the creation and adoption of an eco‐efficiency index for pesticide use so that the broad benefits of eco‐efficient strategies such as IPM can be more readily quantified. We propose an index based upon the ratio of crop yield to a risk quotient (RQ) calculated from pesticide toxicity. Eco‐efficiency is an operational basis for optimizing pest management for sustainability. It naturally favors adoption of IPM and should be considered by regulators, researchers, and practitioners involved in pest management. © 2019 Society of Chemical Industry     

Review and analysis of the potential for glyphosate to interact with the estrogen,androgen and thyroid pathways

Glyphosate was recently evaluated for its potential to interact with the estrogen, androgen and thyroid (EAT) hormone pathways, including steroidogenesis, under the United States Environmental Protection Agency's (USEPA) Endocrine Disruptor Screening Program (EDSP), then by Germany, the rapporteur Member State who led the European Annex 1 renewal for glyphosate, and then by the European Food Protection Agency (EFSA) also as part of the Annex 1 renewal for glyphosate. Under the EDSP, 11 Tier 1 assays were run following the USEPA's validated 890‐series test guidelines and included five in vitro and six in vivo assays to evaluate the EAT pathways. Steroidogenesis was evaluated as part of the estrogen and androgen pathways. An up‐to‐date critical review has been conducted that considered results from the EDSP Tier 1 battery, guideline regulatory studies and an in‐depth analysis of the literature studies that informed an endocrine assessment. A strength of this evaluation was that it included data across multiple levels of biological organization, and mammalian and nonmammalian test systems. There was strong agreement across the in vitro and in vivo Tier 1 battery, guideline studies and relevant literature studies, demonstrating that glyphosate does not interact with EAT pathways including steroidogenesis. Based on an analysis of the comprehensive toxicology database for glyphosate and the literature, this review has concluded that glyphosate does not have endocrine‐disrupting properties through estrogen, androgen, thyroid and steroidogenic modes of action. © 2020 Society of Chemical Industry     

新型氟苯尼考药丸的制备及其在大黄鱼体内的药代动力学分析

大黄鱼养殖业中存在因过量用药而造成环境污染严重及大黄鱼发病率和死亡率高等问题,以淀粉、壳聚糖和明胶三元复合物作为载体,以渔业中应用较多的广谱抗生素氟苯尼考为药物模型,制备了一种可生物降解并可定量给药的抗生素药丸。采用扫描电子显微镜、高效液相色谱法等手段对该药丸的物理特性、吸水性能、抑菌性能及药物残留量进行了检测。结果表明:所制备的载体和药丸具有一致的内部微观结构,多孔结构分布均匀,孔与孔之间具有良好的连通性,有利于药物的流通和释放。体外抑菌试验结果显示,其抑菌能力与氟苯尼考的含量成正相关。经口服药丸后,氟苯尼考在大黄鱼血液、肝脏和肌肉中的药代动力学符合一级吸收二室开放模型的特征,并可在血液、肝脏和肌肉中缓慢消解。     

(Q)SAR tools for the prediction of mutagenic properties: Are they ready for application in pesticide regulation?

The assessment of human health risks resulting from the presence of metabolites in groundwater and food residues has become an important element in pesticide authorisation. In this context, the evaluation of mutagenicity is of particular interest and a paradigm shift from exposure‐triggered testing to in silico‐based screening has been recommended in the European Food Safety Authority (EFSA) Guidance on the establishment of the residue definition for dietary risk assessment. In addition, it is proposed to apply in silico predictions when experimental mutagenicity testing is not possible due to a lack of sufficient quantities of the pesticide metabolite. This, combined with animal welfare and economic considerations, has led to a situation where an increasing number of in silico studies are submitted to regulatory authorities. Whilst there is extensive experience with in silico predictions for mutagenicity in the chemical and pharmaceutical industry, their suitability in pesticide regulation is still insufficiently considered. Therefore, we herein discuss critical issues that need to be resolved to successfully implement (Quantitative) Structure‐Activity Relationship ((Q)SAR) as an accepted tool in pesticide regulation. For illustration purposes, the results of a pilot study are included. The presented study highlights a need for further improvement regarding the predictivity and applicability domain of (Q)SAR systems for pesticides and their metabolites, but also raises other questions such as model selection, establishment of acceptance criteria, harmonised approaches to the combination of model outputs into overall conclusions, adequate reporting and data sharing. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

基于荧光探针技术检测30种农兽药及其二元、三元组合对CYP3A4酶的联合毒性

在农业生产中混合使用多种农药或兽药越来越普遍,但因药剂联合毒性效应的不确定性而对人体健康产生严重威胁。本文基于酶抑制法原理,利用荧光探针 NEN (N-乙基-1,8-萘二甲酰亚胺) 直接检测CYP3A4酶的活性,建立了广谱筛查混合农兽药联合毒性效应的方法,并以常用的30种农兽药及其典型的23种二元和26种三元组合为研究对象,检测了农兽药混合物对CYP3A4酶的联合毒性效应,其中标准质量浓度根据食品安全国家标准规定的农兽药最大残留限量确定。结果表明:3种质量浓度梯度下对CYP3A4酶均具有协同作用的混合农兽药组合有克百威 + 多菌灵、克百威 + 吡虫啉、啶虫脒 + 烯酰吗啉、吡虫啉 + 多菌灵、氯氰菊酯 + 啶虫脒 + 烯酰吗啉、克百威 + 啶虫脒 + 多菌灵、吡虫啉 + 啶虫脒 + 多菌灵、吡虫啉 + 啶虫脒 + 烯酰吗啉、毒死蜱 + 啶虫脒 + 多菌灵和联苯菊酯 + 啶虫脒 + 多菌灵。当单一农兽药对CYP3A4酶活性的抑制率较高时,与其他农兽药混合后联合毒性效应呈拮抗作用,而当单一农兽药对酶活性的抑制率低于2%时,则与其他农兽药混合后联合毒性效应呈现不确定性。农兽药组合在低浓度下对CYP3A4酶的联合毒性往往存在较强的协同作用,但随着浓度的升高,联合毒性效应从协同变为拮抗作用。分析农兽药与CYP3A4酶之间的构效关系可知,含有芳氯基团的数量与对酶活性的抑制程度成正相关,含有3个芳氯及以上基团的农兽药对CYP3A4酶活性的抑制作用最为显著,抑制率在30%以上,如百菌清、毒死蜱、甲基毒死蜱、咪鲜胺等;含有2个芳氯或“强吸电子基团 + 1个芳氯”基团的农兽药,对CYP3A4酶活性的抑制作用较强,抑制率在18%以上,如苯醚甲环唑、哒螨灵和异菌脲等。具有氨基甲酸酯基团的农兽药单独作用于CYP3A4酶毒性较小或几乎没有毒性时,与其他农兽药混合后显示较强的协同作用。本研究建立的检测方法为广谱筛查混合农兽药联合毒性提供了新思路,检测结果可为进一步在细胞和动物水平制订农兽药混剂的风险评估方案提供依据。     

Rapid in vitro screening assay for immunotoxic effects of organophosphorus and carbamate insecticides on the generation of cytotoxic T-lymphocyte responses

There has been an increasing need for rapid and easily interpreted techniques for the screening of possible immunotoxicants. Besides the obvious detrimental effects of exposure to immunosuppressive agents, the modulation of the immune system which results from exposure to these toxicants may be a sensitive index to the toxicologic effects of such agents. Other researchers have proposed assays to screen the effect of in vitro treatment with immunotoxicants on mitogenic and humoral immune responses. In this report, we have described an in vitro technique for screening the effect of immunotoxicants, in the presence and absence of a NADPH fortified liver postmitochondrial supernatant (S-9) from Arochlor 1254-treated rat, on another aspect of the mammalian immune system, the generation of a T-cell-mediated cytolytic (CTL) response. This enzyme system altered the effect of organophosphorus compounds on the generation of a CTL response. Malathion and fenitrothion were no longer suppressive following this pretreatment; however, ethyl and methyl parathion and fenthion were only partially detoxified. In contrast, the S-9 enzyme system did not alter the effect of carbamate pesticides, carbaryl and carbofuran, on the generation of CTL responses. This report describes the effects of these seven organophosphorus and carbamate pesticides on the generation of the CTL response. In addition, some of the in vivo data published on the immunomodulatory effects of these compounds were collated from the literature and a correlation between in vitro and in vivo studies was discussed.     

Some factors affecting the activities of dinitrophenol fungicides

The vapour phase and protectant activities of members of seven homologous series of alkyldinitrophenols against cucumber powdery mildew (Oidium sp.) are discussed. No correlation existed between vapour activities in vitro or in vivo and vapour pressure, but a positive correlation between Vapour' protection in vivo and conventional protectant activity was evident. Although vapour activity occurs with some homologues, results obtained for zone assays in vivo are probably better explained in terms of easy movement of the compound in the leaf surface than in terms of vapour transmission. Alkyldinitrophenyl-crotonates showed no zone activity in vivo but often good protectant activity. The possible existence of two optimum π-values for protectant activity is suggested. No appreciable systemic activity was found with these compounds.     

Entomopathogenic nematodes for the management of Agrotis ipsilon: effect of instar, nematode species and nematode production method

BACKGROUND: Previous laboratory studies have indicated the potential of some entomopathogenic nematode (EPN) species for the control of larvae of the black cutworm (BCW). To determine the most promising EPN species and the most susceptible BCW stages, a more in‐depth evaluation of seven EPN species against different BCW instars was carried out, the efficacies of in vitro‐ and in vivo‐produced EPNs were compared and the suitability of BCW instars for EPN reproduction was examined. RESULTS: Heterorhabditis megidis was the most virulent species, irrespective of larval stage in small arenas, followed most often by H. bacteriophora. In pots with grass, Steinernema carpocapsae tended to be the most virulent species, followed by H. bacteriophora, H. megidis and S. riobrave. Fourth and/or fifth instars were the most susceptible stages to most EPN species, and pupae the least susceptible. Furthermore, H. bacteriophora, H. megidis and S. carpocapsae successfully reproduced in fifth and sixth instars and pupae. In vivo‐produced H. megidis and S. carpocapsae controlled fifth instars better than the corresponding in vitro‐produced products; production method did not affect H. bacteriophora and S. riobrave efficacy. CONCLUSIONS: Several in vitro‐produced commercial EPN strains were highly virulent to BCW and warrant further testing under field conditions, along with some in vivo‐produced strains. Copyright © 2011 Society of Chemical Industry     

六种农药对两种果蝇成虫室内毒力和主要解毒酶活性的影响

为明确田间常用农药对伊米果蝇Drosophila immigrans和海德氏果蝇D. hydei的毒力和解毒机制,采用药膜法在室内测定6种田间常用农药原药对2种果蝇实验种群成虫的LC_(10)、LC_(20)和LC_(50),并研究其中3种农药的亚致死剂量(LC_(10)、LC_(20))对果蝇谷胱甘肽S-转移酶(GST)、羧酸酯酶(CarE)、乙酰胆碱酯酶(AchE)3种主要解毒酶活性的影响。结果表明,乙基多杀菌素对伊米果蝇的毒力最大,LC_(10)、LC_(20)和LC_(50)分别为0.29、0.51和1.51 mg/L;甲维盐对海德氏果蝇的毒力最大,LC_(10)、LC_(20)和LC_(50)分别为0.14、0.36和2.09 mg/L;吡虫啉对2种果蝇的毒力均最低。不同亚致死浓度的乙基多杀菌素处理伊米果蝇24 h后,CarE和AchE活性均显著高于对照,而GST活性在低浓度时显著高于对照;高浓度甲维盐仅对海德氏果蝇AchE活性有显著抑制作用;吡虫啉可抑制伊米果蝇AchE和海德氏果蝇CarE活性。表明伊米果蝇和海德氏果蝇可通过改变3种解毒酶的活性来防御杀虫剂对其造成的影响。     

Can the mammalian organoid technology be applied to the insect gut?

Mammalian intestinal organoids are multicellular structures that closely resemble the structure of the intestinal epithelium and can be generated in vitro from intestinal stem cells under appropriate culture conditions. This technology has transformed pharmaceutical research and drug development in human medicine. For the insect gut, no biotechnological platform equivalent to organoid cultures has been described yet. Comparison of the regulation of intestinal homeostasis and growth between insects and mammals has revealed significant similarities but also important differences. In contrast to mammals, the differentiation potential of available insect cell lines is limited and can not be exploited for in vitro permeability assays to measure the uptake of insecticides. The successful development of in vitro models could be a result of the emergence of molecular mechanisms of self‐organization and signaling in the intestine that are unique to mammals. It is nevertheless considered that the technology gap is a consequence of vast differences in knowledge, particularly with respect to culture conditions that maintain the differentation potential of insect midgut cells. From the viewpoint of pest control, advanced in vitro models of the insect midgut would be very desirable because of its key barrier function for orally ingested insecticides with hemolymphatic target and its role in insecticide resistance. © 2020 Society of Chemical Industry