A Decision Logic for the Reliability Assessment and Interpretation of Vitellogenin Measurements

IF 3.6 4区 环境科学与生态学 Q2 ENVIRONMENTAL SCIENCES Environmental Toxicology and Chemistry Pub Date : 2024-07-09 DOI:10.1002/etc.5946
Rebecca J. Brown, Grace H. Panter, Natalie Burden, Lennart Weltje, James R. Wheeler, Edward R. Salinas, Yvonne Wolf, Laurent Lagadic
{"title":"A Decision Logic for the Reliability Assessment and Interpretation of Vitellogenin Measurements","authors":"Rebecca J. Brown,&nbsp;Grace H. Panter,&nbsp;Natalie Burden,&nbsp;Lennart Weltje,&nbsp;James R. Wheeler,&nbsp;Edward R. Salinas,&nbsp;Yvonne Wolf,&nbsp;Laurent Lagadic","doi":"10.1002/etc.5946","DOIUrl":null,"url":null,"abstract":"<p>The egg-yolk precursor protein vitellogenin (VTG) is a biomarker for the determination of in vivo endocrine activity of chemicals in animals. Measurements of VTG in fish and amphibians are included in Organisation for Economic Co-operation and Development (OECD) test guidelines to provide support for identifying potential endocrine-active substances acting on the estrogen, androgen, and steroidogenesis (EAS) pathways. Induction of VTG in male fish is often associated with estrogenic activity, whereas inhibition in female fish may be related to substances that inhibit estrogen synthesis. The VTG protein or mRNA is measured in the plasma, liver, or whole body of fish, depending on the species and developmental stage, and on the specific test guideline requirements. Concerns have been raised regarding the variability of VTG measurements in fish, which could challenge the reliability and acceptability of VTG results for use in regulatory assessment of chemicals (Brown et al., <span>2023</span>). Hence, it is important to correctly measure and interpret VTG results, because ambiguous effects could trigger additional, potentially unnecessary, higher tier testing (i.e., animal-intensive life-cycle studies) to confirm or refute the VTG result. A literature review of VTG data from standard fish species exposed to 106 substances showed high intra- and interlaboratory variability in VTG concentrations, as well as discrepancies in the interpretation of results based on large differences between fish held in dilution water versus solvent controls, or due to the presence of outlier measurements (Brown et al., <span>2023</span>). For instance, the coefficient of variation of VTG concentrations in control adult fathead minnows (<i>Pimephales promelas</i>) was 543.2% for males and 206.1% for females. The same review also found evidence for false-positive/negative responses and situations in which the VTG results were difficult to interpret.</p><p>Findings from a laboratory survey to help understand the sources of variability in VTG protein and mRNA measurements identified three areas for improvement and harmonization: (1) sampling and storage, (2) quantification, and (3) data handling and statistical analysis (Burden et al., <span>2023</span>).</p><p>The survey also highlighted a need for the development of a decision logic to assist in the acceptability, determination, and interpretation of VTG measurements. This would support the development of new OECD guidance detailing best practice for VTG methodology, applicable across relevant test guideline studies but also applicable to studies published in the open literature. In the proposed decision logic (Figure 1), the reliability of the VTG results is assessed separately from the overall study reliability, because a reliable study (as evaluated against, for example, the Klimisch criteria [Klimisch et al., <span>1997</span>] or the Criteria for Reporting and Evaluating Ecotoxicity Data [Moermond et al., <span>2016</span>]), may still include unreliable VTG analyses. The reliability of VTG results should therefore be based on specific quality criteria (e.g., as outlined in OECD [<span>2012</span>] test guideline 229: Fish short term reproduction assay, and summarized in Burden et al., <span>2023</span>). A lack of reporting key information such as the calibration curve statistics, or limits of detection (LoD) or limits of quantification (LoQ) can make it difficult to assess the reliability of the VTG result.</p><p>Data handling and statistical analysis require special consideration as part of OECD guidance on VTG methodology and interpretation (Burden et al., <span>2023</span>)—from the survey it was evident that laboratories have different approaches. This includes the value used to represent male VTG concentrations when they are below the LoQ (e.g., the LoQ value itself, half the LoQ value, the LoD value or zero), or the identification and handling of outlier VTG values. Outliers can be identified as values that exceed the median plus three times the interquartile range (i.e., the difference between the 75th and 25th percentiles) or via formal outlier tests (e.g., Grubb's test). Conducting statistical analysis with and without outliers is the most transparent way of presenting the data. In addition to general recommendations for the statistical analysis of VTG data based on OECD test guideline 54 (OECD, <span>2006</span>), methods addressing specific aspects of VTG data handling should be more explicit in the proposed new guidance (i.e., related to hypothesis testing when the sex-related response is one sided or the value to use in case male VTG is below the LoQ).</p><p>According to the European Chemicals Agency (ECHA) and European Food Safety Authority (EFSA) guidance for the identification of endocrine disruptors (ECHA/EFSA, <span>2018</span>), a decrease in VTG may also be caused by overt or systemic toxicity, nonendocrine mechanisms (e.g., hepatotoxicity), or confounding factors such as diet or infection (Dang, <span>2016</span>), and should not necessarily be interpreted as being due to an endocrine mechanism. When one is considering whether inhibition of VTG is endocrine mediated, the effect should be interpreted in combination with other observations including systemic toxicity (typically based on an arbitrary threshold of 10% mortality related to the definition of the Maximum Tolerated Concentration) and other overt signs of nonlethal toxicity, such as behavioral changes (e.g., loss of equilibrium, lethargy), gill respiration rate, reduced growth, discoloration, and reduced feeding (Hutchinson et al., <span>2009</span>; Wheeler et al., <span>2013</span>).</p><p>The existence and shape of the concentration–response and the magnitude of VTG effects in exposed organisms compared with controls are also important considerations (e.g., responses should be considered in the context of concentration–response relationships to ensure biological relevance). This is especially the case for screening studies, which often use a wide spacing (up to 10-fold) between test concentrations. Based on the literature review, small changes in VTG (e.g., &lt;100% induction in males and &lt;30% induction or inhibition in females for <i>P. promelas</i>) are unlikely to be biologically relevant as indicators for endocrine activity, considering experimental and species-specific variability (Brown et al., <span>2023</span>). The consistency of VTG changes should therefore be considered alongside other endocrine-mediated endpoints such as specific gonadal histopathology findings (Ankley &amp; Jensen, <span>2014</span>).</p><p>Finally, VTG changes should be checked for consistency with other available data (i.e., in silico, in vitro, in vivo studies in mammals and knowledge of robust adverse outcome pathways). In utilizing all available data in a Weight of Evidence (WoE) analysis, it may be possible to confirm or refute whether there is a need for additional higher tier fish testing. The WoE can also be applied where there are conflicting VTG results, for example, by giving more weight to studies that have well-reported VTG analysis and meet the data quality criteria. A WoE could also be used in determining the next steps following an equivocal VTG result. For example, an equivocal effect on male VTG, in the absence of other evidence for endocrine activity, could potentially be resolved by conducting relevant in vitro assays covering the EAS pathways (e.g., with the estrogen receptor transactivation assay [OECD, <span>2021a</span>], the aromatase inhibition assay [US Environmental Protection Agency, <span>2009</span>], or eleutheroembryonic (nonprotected life stage) assays, such as the EASZY assay [OECD, <span>2021b</span>] or the RADAR assay [OECD, <span>2022</span>]). Such a mechanistic approach may avoid conducting animal studies or justify a move directly to higher tier animal testing.</p><p>In conclusion, the proposed decision logic provides a basis for more consistent and transparent VTG measurement and reporting and will assist end users, including regulatory scientists, in interpreting the data. The overall aim is to support the development of OECD guidance on best practice for VTG assessment in aquatic vertebrates.</p><p><b>Rebecca Jayne Brown</b>: Conceptualization; Writing—original draft; Writing—review &amp; editing. <b>Grace Panter</b>: Conceptualization; Writing—original draft; Writing—review &amp; editing. <b>Natalie Burden</b>: Conceptualization; Writing—original draft; Writing—review &amp; editing. <b>Lennart Weltje</b>: Conceptualization; Writing—original draft; Writing—review &amp; editing. <b>James Wheeler</b>: Conceptualization; Writing—original draft; Writing—review &amp; editing. <b>Edward Salinas</b>: Conceptualization; Writing—original draft; Writing—review &amp; editing. <b>Yvonne Wolf</b>: Conceptualization; Writing—original draft; Writing—review &amp; editing. <b>Laurent Lagadic</b>: Conceptualization; Writing—original draft; Writing—review &amp; editing.</p>","PeriodicalId":11793,"journal":{"name":"Environmental Toxicology and Chemistry","volume":null,"pages":null},"PeriodicalIF":3.6000,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/etc.5946","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Environmental Toxicology and Chemistry","FirstCategoryId":"93","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/etc.5946","RegionNum":4,"RegionCategory":"环境科学与生态学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ENVIRONMENTAL SCIENCES","Score":null,"Total":0}
引用次数: 0

Abstract

The egg-yolk precursor protein vitellogenin (VTG) is a biomarker for the determination of in vivo endocrine activity of chemicals in animals. Measurements of VTG in fish and amphibians are included in Organisation for Economic Co-operation and Development (OECD) test guidelines to provide support for identifying potential endocrine-active substances acting on the estrogen, androgen, and steroidogenesis (EAS) pathways. Induction of VTG in male fish is often associated with estrogenic activity, whereas inhibition in female fish may be related to substances that inhibit estrogen synthesis. The VTG protein or mRNA is measured in the plasma, liver, or whole body of fish, depending on the species and developmental stage, and on the specific test guideline requirements. Concerns have been raised regarding the variability of VTG measurements in fish, which could challenge the reliability and acceptability of VTG results for use in regulatory assessment of chemicals (Brown et al., 2023). Hence, it is important to correctly measure and interpret VTG results, because ambiguous effects could trigger additional, potentially unnecessary, higher tier testing (i.e., animal-intensive life-cycle studies) to confirm or refute the VTG result. A literature review of VTG data from standard fish species exposed to 106 substances showed high intra- and interlaboratory variability in VTG concentrations, as well as discrepancies in the interpretation of results based on large differences between fish held in dilution water versus solvent controls, or due to the presence of outlier measurements (Brown et al., 2023). For instance, the coefficient of variation of VTG concentrations in control adult fathead minnows (Pimephales promelas) was 543.2% for males and 206.1% for females. The same review also found evidence for false-positive/negative responses and situations in which the VTG results were difficult to interpret.

Findings from a laboratory survey to help understand the sources of variability in VTG protein and mRNA measurements identified three areas for improvement and harmonization: (1) sampling and storage, (2) quantification, and (3) data handling and statistical analysis (Burden et al., 2023).

The survey also highlighted a need for the development of a decision logic to assist in the acceptability, determination, and interpretation of VTG measurements. This would support the development of new OECD guidance detailing best practice for VTG methodology, applicable across relevant test guideline studies but also applicable to studies published in the open literature. In the proposed decision logic (Figure 1), the reliability of the VTG results is assessed separately from the overall study reliability, because a reliable study (as evaluated against, for example, the Klimisch criteria [Klimisch et al., 1997] or the Criteria for Reporting and Evaluating Ecotoxicity Data [Moermond et al., 2016]), may still include unreliable VTG analyses. The reliability of VTG results should therefore be based on specific quality criteria (e.g., as outlined in OECD [2012] test guideline 229: Fish short term reproduction assay, and summarized in Burden et al., 2023). A lack of reporting key information such as the calibration curve statistics, or limits of detection (LoD) or limits of quantification (LoQ) can make it difficult to assess the reliability of the VTG result.

Data handling and statistical analysis require special consideration as part of OECD guidance on VTG methodology and interpretation (Burden et al., 2023)—from the survey it was evident that laboratories have different approaches. This includes the value used to represent male VTG concentrations when they are below the LoQ (e.g., the LoQ value itself, half the LoQ value, the LoD value or zero), or the identification and handling of outlier VTG values. Outliers can be identified as values that exceed the median plus three times the interquartile range (i.e., the difference between the 75th and 25th percentiles) or via formal outlier tests (e.g., Grubb's test). Conducting statistical analysis with and without outliers is the most transparent way of presenting the data. In addition to general recommendations for the statistical analysis of VTG data based on OECD test guideline 54 (OECD, 2006), methods addressing specific aspects of VTG data handling should be more explicit in the proposed new guidance (i.e., related to hypothesis testing when the sex-related response is one sided or the value to use in case male VTG is below the LoQ).

According to the European Chemicals Agency (ECHA) and European Food Safety Authority (EFSA) guidance for the identification of endocrine disruptors (ECHA/EFSA, 2018), a decrease in VTG may also be caused by overt or systemic toxicity, nonendocrine mechanisms (e.g., hepatotoxicity), or confounding factors such as diet or infection (Dang, 2016), and should not necessarily be interpreted as being due to an endocrine mechanism. When one is considering whether inhibition of VTG is endocrine mediated, the effect should be interpreted in combination with other observations including systemic toxicity (typically based on an arbitrary threshold of 10% mortality related to the definition of the Maximum Tolerated Concentration) and other overt signs of nonlethal toxicity, such as behavioral changes (e.g., loss of equilibrium, lethargy), gill respiration rate, reduced growth, discoloration, and reduced feeding (Hutchinson et al., 2009; Wheeler et al., 2013).

The existence and shape of the concentration–response and the magnitude of VTG effects in exposed organisms compared with controls are also important considerations (e.g., responses should be considered in the context of concentration–response relationships to ensure biological relevance). This is especially the case for screening studies, which often use a wide spacing (up to 10-fold) between test concentrations. Based on the literature review, small changes in VTG (e.g., <100% induction in males and <30% induction or inhibition in females for P. promelas) are unlikely to be biologically relevant as indicators for endocrine activity, considering experimental and species-specific variability (Brown et al., 2023). The consistency of VTG changes should therefore be considered alongside other endocrine-mediated endpoints such as specific gonadal histopathology findings (Ankley & Jensen, 2014).

Finally, VTG changes should be checked for consistency with other available data (i.e., in silico, in vitro, in vivo studies in mammals and knowledge of robust adverse outcome pathways). In utilizing all available data in a Weight of Evidence (WoE) analysis, it may be possible to confirm or refute whether there is a need for additional higher tier fish testing. The WoE can also be applied where there are conflicting VTG results, for example, by giving more weight to studies that have well-reported VTG analysis and meet the data quality criteria. A WoE could also be used in determining the next steps following an equivocal VTG result. For example, an equivocal effect on male VTG, in the absence of other evidence for endocrine activity, could potentially be resolved by conducting relevant in vitro assays covering the EAS pathways (e.g., with the estrogen receptor transactivation assay [OECD, 2021a], the aromatase inhibition assay [US Environmental Protection Agency, 2009], or eleutheroembryonic (nonprotected life stage) assays, such as the EASZY assay [OECD, 2021b] or the RADAR assay [OECD, 2022]). Such a mechanistic approach may avoid conducting animal studies or justify a move directly to higher tier animal testing.

In conclusion, the proposed decision logic provides a basis for more consistent and transparent VTG measurement and reporting and will assist end users, including regulatory scientists, in interpreting the data. The overall aim is to support the development of OECD guidance on best practice for VTG assessment in aquatic vertebrates.

Rebecca Jayne Brown: Conceptualization; Writing—original draft; Writing—review & editing. Grace Panter: Conceptualization; Writing—original draft; Writing—review & editing. Natalie Burden: Conceptualization; Writing—original draft; Writing—review & editing. Lennart Weltje: Conceptualization; Writing—original draft; Writing—review & editing. James Wheeler: Conceptualization; Writing—original draft; Writing—review & editing. Edward Salinas: Conceptualization; Writing—original draft; Writing—review & editing. Yvonne Wolf: Conceptualization; Writing—original draft; Writing—review & editing. Laurent Lagadic: Conceptualization; Writing—original draft; Writing—review & editing.

Abstract Image

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
卵黄素测量可靠性评估和解释的决策逻辑。
在考虑 VTG 的抑制作用是否由内分泌介导时,应结合其他观察结果来解释该效应,包括系统毒性(通常基于与最大耐受浓度定义相关的 10%死亡率的任意阈值)和其他非致命毒性的明显迹象,如行为变化(如失去平衡、嗜睡、鳃呼吸速率、生长减少、褪色和摄食减少)(Hutchinson 等人,2009 年;Wheeler 等人,2013 年)、与对照组相比,暴露生物的浓度-反应和 VTG 影响的存在和形状也是重要的考虑因素(例如,应在浓度-反应关系的背景下考虑反应,以确保生物相关性)。对于筛选研究来说,尤其如此,因为筛选研究通常会在试验浓度之间使用较大的间隔(最多 10 倍)。根据文献综述,考虑到实验和物种的特定变异性,VTG 的微小变化(例如,对海马来说,雄性 100% 的诱导和雌性 30% 的诱导或抑制)不太可能作为内分泌活性的生物相关指标(Brown 等人,2023 年)。因此,VTG 变化的一致性应与其他内分泌介导的终点(如特定的性腺组织病理学发现)一起考虑(Ankley &amp; Jensen, 2014)。最后,应检查 VTG 变化与其他可用数据(即哺乳动物体内、体外和体内研究的硅学数据,以及关于可靠的不良结果途径的知识)的一致性。在证据权重(WoE)分析中利用所有可用数据,可以确认或反驳是否需要进行更高级别的鱼类试验。当出现相互矛盾的 VTG 结果时,也可采用 WoE,例如,对那些报告了充分的 VTG 分析且符合数据质量标准的研究给予更多权重。在 VTG 结果不明确的情况下,也可以使用 WoE 来决定下一步的行动。例如,在没有其他内分泌活性证据的情况下,如果对男性 VTG 的影响模棱两可,则可通过进行涵盖 EAS 途径的相关体外试验(如雌激素受体转录)来解决、雌激素受体反式激活试验[经合组织,2021a]、芳香化酶抑制试验[美国环境保护局,2009 年]或榄香烯胚胎(非保护生命阶段)试验,如 EASZY 试验[经合组织,2021b]或 RADAR 试验[经合组织,2022])。总之,拟议的决策逻辑为更一致、更透明的 VTG 测量和报告提供了基础,并将帮助包括监管科学家在内的最终用户解释数据。总体目标是支持经合组织制定水生脊椎动物 VTG 评估最佳实践指南:构思;撰写-初稿;撰写-审核;编辑。格蕾丝-潘特构思;撰写-原稿;撰写-审核和编辑。娜塔莉-伯登概念化;写作-原稿;写作-审核与编辑。伦纳特-韦尔杰概念化;写作-原稿;写作-审核和编辑。詹姆斯-惠勒构思;撰写-原稿;撰写-审核和编辑。爱德华-萨利纳斯构思;撰写-原稿;撰写-审核和编辑。伊冯娜-沃尔夫构思;写作-原稿;写作-审核和编辑。Laurent Lagadic:概念化;写作-原稿;写作-审核和编辑。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
CiteScore
7.40
自引率
9.80%
发文量
265
审稿时长
3.4 months
期刊介绍: The Society of Environmental Toxicology and Chemistry (SETAC) publishes two journals: Environmental Toxicology and Chemistry (ET&C) and Integrated Environmental Assessment and Management (IEAM). Environmental Toxicology and Chemistry is dedicated to furthering scientific knowledge and disseminating information on environmental toxicology and chemistry, including the application of these sciences to risk assessment.[...] Environmental Toxicology and Chemistry is interdisciplinary in scope and integrates the fields of environmental toxicology; environmental, analytical, and molecular chemistry; ecology; physiology; biochemistry; microbiology; genetics; genomics; environmental engineering; chemical, environmental, and biological modeling; epidemiology; and earth sciences. ET&C seeks to publish papers describing original experimental or theoretical work that significantly advances understanding in the area of environmental toxicology, environmental chemistry and hazard/risk assessment. Emphasis is given to papers that enhance capabilities for the prediction, measurement, and assessment of the fate and effects of chemicals in the environment, rather than simply providing additional data. The scientific impact of papers is judged in terms of the breadth and depth of the findings and the expected influence on existing or future scientific practice. Methodological papers must make clear not only how the work differs from existing practice, but the significance of these differences to the field. Site-based research or monitoring must have regional or global implications beyond the particular site, such as evaluating processes, mechanisms, or theory under a natural environmental setting.
期刊最新文献
Correction. Spotlights are papers selected by editors published in peer-reviewed journals that may be more regionally specific or appearing in languages other than English Issue Information - Cover Editorial Board and Table of Contents Detection and Prediction of Toxic Aluminum Concentrations in High-Priority Salmon Rivers in Nova Scotia.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1