Incorporation of metabolic enzymes to improve predictivity of reporter gene assay results for estrogenic and anti-androgenic activity

Vugt-Lussenburg, Barbara M.A. van; Lee, Rosan B. van der; Man, Hai Yen; Middelhof, Irene; Brouwer, Abraham; Besselink, Harrie; Burg, Bart van der

doi:10.1016/j.reprotox.2017.11.005

Cited by 31 publications

(13 citation statements)

References 33 publications

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“…In the AR-CALUX ® cell line, a low metabolic activity was shown via RNA sequencing where major classes of metabolic genes were targeted and found to have no or low expression (personal communication from BDS). By incorporating S9 fraction into the method, the impact of metabolism on test chemical activity can be studied ( van Vugt-Lussenburg et al , 2018 ). This is the subject of an ongoing study.…”

Section: Discussionmentioning

confidence: 99%

Reliability of the AR-CALUX®In Vitro Method Used to Detect Chemicals with (Anti)Androgen Activity: Results of an International Ring Trial

Milcamps

Liška

Langezaal

et al. 2021

Toxicological Sciences

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The AR-CALUX® in vitro method is a reporter gene-based transactivation method where endocrine active chemicals with androgenic or anti-androgenic potential can be detected. Its primary purpose is for screening chemicals for further prioritisation and providing mechanistic (endocrine mode of action) information, as defined by the OECD Conceptual Framework for the testing and assessment of endocrine disrupting chemicals. This paper describes the conduct and results of an international ring trial with three EU-NETVAL laboratories and the test method developer. It was organised by EURL ECVAM to validate the method by testing 46 chemicals. A very good reproducibility within and between laboratories was concluded (94.7 – 100% and 100% concordance of classification) with low within and between laboratory variability (less than 2.5% CV on EC50 values). Moreover, the variability is within the range of other validated, mechanistically similar methods. In comparison to the AR-reference list compiled by ICCVAM, an almost 100% concordance of classifications was obtained. This method allows the detection of agonist and antagonist properties of a chemical. A specificity control test was developed during the validation study and added to the antagonist assay rendering the assay more specific. A comparison is made with the mechanistically similar methods AR-EcoScreen™ and 22Rv1/MMTV GR-KO TA. The AR-CALUX® method was approved for inclusion in the recently updated OECD test guideline TG458 which incorporates all three methods.

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Section: Discussionmentioning

confidence: 99%

Reliability of the AR-CALUX®In Vitro Method Used to Detect Chemicals with (Anti)Androgen Activity: Results of an International Ring Trial

Milcamps

Liška

Langezaal

et al. 2021

Toxicological Sciences

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“…For example, BPA and genistein showed lower estrogenic activities after phase II metabolism. In contrast, after phase I metabolism, 5 of the chemicals showed lower estrogenic activities, and 11 of the chemicals showed higher estrogenic activities [73].…”

Section: Testing Strategiesmentioning

confidence: 90%

“…In a recent study, a rat S9 fraction was incorporated in a CALUX reporter gene assay (i.e., the U2-OS cell line), and 27 chemicals were evaluated [73]. Selective inclusion of cofactors enables the evaluation of the different phases of metabolism on the estrogenic activities (i.e., phase I, nicotinamide adenine dinucleotide phosphate [reduced]; phases I and II, nicotinamide adenine dinucleotide phosphate [reduced], UDP-glucuronic acid, phosphoadenosylphosphosulfate, glutathione).…”

Section: Testing Strategiesmentioning

confidence: 99%

The Role of Metabolism in the Estrogenic Activity of Endocrine-Disrupting Chemicals

Skledar¹,

Mašič²

2019

Estrogen

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Exposure to several natural and synthetic chemicals can disrupt the endocrine system and thus present a threat to human health. In vivo, such chemicals can be metabolized, which can change the endocrine activity of the parent chemical. Metabolism is usually considered to be a detoxification process, as it generally appears to reduce the estrogenic activity of a chemical and accelerate its elimination from the body. This is seen for bisphenol A (BPA), a known agonist of the estrogen receptor, whereby BPA glucuronide has no effects on this receptor. In contrast, numerous metabolites that show significantly greater estrogenic activities from their parent chemicals have been described in the literature. An example is the ipso metabolite of BPA, 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene, which shows >100-fold estrogenic activity compared to BPA. Consideration of metabolic pathways in in vitro models is therefore of great importance for reliable analysis and correct in vitro to in vivo correlations. The inclusion of metabolic aspects in these assays will reduce false-positive data for chemicals that are detoxified in vivo and false-negative data for proestrogens. Different approaches for this incorporation of metabolic systems for determination of estrogenic activities are already in use and are described in the present chapter.

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“…U-2 OS cells: These osteosarcoma cells are relatively de-differentiated and grow in an epitheloid way. Their viability was assessed based on constitutive luciferase expression (van Vugt-Lussenburg et al 2018 ) in the context of the automated CALUX ® reporter gene assay procedure (see paragraph below). A detailed SOP is available at the ECVAM DB-ALM database (protocol No.…”

Section: Methodsmentioning

confidence: 99%

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods

et al. 2020

Self Cite

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Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

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Incorporation of metabolic enzymes to improve predictivity of reporter gene assay results for estrogenic and anti-androgenic activity

Cited by 31 publications

References 33 publications

Reliability of the AR-CALUX®In Vitro Method Used to Detect Chemicals with (Anti)Androgen Activity: Results of an International Ring Trial

Reliability of the AR-CALUX®In Vitro Method Used to Detect Chemicals with (Anti)Androgen Activity: Results of an International Ring Trial

The Role of Metabolism in the Estrogenic Activity of Endocrine-Disrupting Chemicals

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods

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