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    Biochemical Ecotoxicology: Principles and Methods
    Biochemical Ecotoxicology: Principles and Methods
    Biochemical Ecotoxicology: Principles and Methods
    Ebook500 pages4 hours

    Biochemical Ecotoxicology: Principles and Methods

    By Francois Gagne

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    About this ebook

    Biochemical Ecotoxicology: Principles and Methods presents practical approaches to biochemical ecotoxicology experiments for environmental protection and conservation.

    With its methodical, stepped approach this essential reference introduces readers to current techniques for toxicity endpoint testing, suitable for laboratories of any size and budget. Each chapter presents a state-of-the-art principle, a quick and inexpensive procedure (including appropriate reagents), case studies, and demonstrations on how to analyze your results.

    Generic techniques are covered, suitable for a variety of organisms, as well as high-throughput techniques like quantitative polymerase chain reactions and enzyme-linked immunoassays. Cutting-edge approaches, including gPCR arrays and lipidomic techniques, are also included, making this is an essential reference for anyone who needs to assess environmental toxicity.

    • Practical, cost-effective approaches to assess environmental toxicity endpoints for all types of organism
    • Presents theory, methods, case studies and information on how to analyze results
    • State-of-the-art techniques, such as ‘omics’ approaches to toxicology
    LanguageEnglish
    PublisherAcademic Press
    Release dateJul 7, 2014
    ISBN9780124116238
    Biochemical Ecotoxicology: Principles and Methods

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      Book preview

      Biochemical Ecotoxicology - Francois Gagne

      2002;132:483–492.

      Chapter 1

      Quantitative Assessments of Biochemical Analyses

      François Gagné

      Quantitative analysis of biomarkers based on biochemical assays requires a number of criteria to ensure the quality of the data. The basic criteria are replication at the analytical and biological levels, calibration, normalization, and the use of reference materials. Analytical methods and biological responses are subject to variations that need to be determined in any science project. The variation of the analytical methodology should be, of course, as low as possible and below the biological variation range (interindividual variation) of the biomarker. To achieve this, optimal replication is essential at both the analytical and biomarker response (individual) levels. For example, the number of replications at the analytical level of an analytical method with 0.5–2% variation would be less than an analytical method with 5–10% variation. This (replication) is in keeping with the theoretical limit of detection, which is defined as a value >2× the SD of the blank. In this respect, replication is essential to ensure reproducibility of the methodology and reliability of the biomarker measurement. The analytical methodology is usually calibrated with a prepared standard of the analyte. The standard could be either external (i.e., pure preparation in the assay blank) or internal (i.e., known amount of standard is added in the sample) depending on the specificity of the method and susceptibility to matrix effects of the (biologically complex) test sample. Usually biomarkers are normalized against biomass (g tissue wet or dry weight, total protein levels) by simply dividing the biomarker with biomass metric. This normalization usually works when the biomarker signal is very low (<0.1%) in respect to the biomass metric and if the ratio (slope) between the biomarker and biomass is constant across the test samples. In a situation where the slope of the biomarker and biomass changes (and remains significant), the normalization should be done using the residual extraction method. When the biomarker represents a significant proportion of the biomass (>0.1%), of the biomass, normalization should be done by one of the following procedures: (1) normalize against a larger biomass unit (dry or wet weight of tissue), (2) use the residual extraction method if correlation remains between the analyte level and biomass unit, and (3) by a novel and robust stepwise addition approach. Finally, the use of reference materials should be used to determine the methodological trueness of the assay, especially when the biochemical assessments are performed on a long-term basis or performed in separate batches at different times or in different laboratories and instruments. Ideally, commercially available certified reference materials should be used when available or prepared in-house at the beginning of the project.

      Keywords

      calibration; detection limit; matrix effects; normalization methods reference materials; replication; reproducibility

      Chapter Outline

      1.1 General Overview 1

      1.2 Replication in Data Analysis 5

      1.3 Replication of the Experiment 6

      1.3.1 Calibration or Standardization 7

      1.3.2 Using Specific and Generic Standards 8

      1.4 Reference Substances 10

      1.5 Defining Detection Limits in the Absence of Analytical Standards 10

      1.6 Reproducibility 12

      1.6.1 Reproducibility at the Calibration Level 12

      1.6.2 Reproducibility at the Level of the Experiment 13

      1.7 Using Reference Material to Standardize Methodologies 13

      1.8 Normalization Approaches 15

      1.8.1 Residual Extraction Method 16

      1.8.2 Stepwise Addition Method 18

      References 19

      1.1 General Overview

      In ecotoxicological studies, the early biological (toxic) effects of chemicals are determined by the use of biochemical markers. Biomarkers rely on chemical analyses of biological macromolecules such as deoxyribonucleic acids, polypeptides/proteins, and membrane assemblages. The ability of proteins to catalyze biochemical reactions (enzymes) is noteworthy and constitutes an important part of quantitative biochemistry. The chemical analyses are identical to quantitative analysis of chemicals in the classic sense (i.e., evaluation of mercury by cold vapor atomic absorption spectrometry) but differ because the analysis is done in relation to a given biological function that often involves highly complex molecules (i.e., mercury binding thiol-rich proteins such as metallothioneins, MTs). As another example, the quantitative evaluation of serotonin, a derivative of the amino acid tryptophan, by liquid chromatography with electrochemical detection is an example of chemical analysis, but the interaction of serotonin on membrane receptors in nerve cell membrane is biochemical because it intervenes in complex biochemical interactions with macromolecules. These assays are practiced in the context of hypothesis testing, i.e., in studies to determine negative interactions, if any, of exposure to a given xenobiotic to a physiological function in the exposed organisms or cells. Moreover, test organisms in the real world are genetically heterogeneous and under various habitat pressures such as nutritional stress, temperature, climate variations, and reproductive cycle. This brings more interindividual variability in the measures [1]. This variation is added to the variation of the method of biochemical analysis. It is therefore essential to determine both the variation induced by the method of analysis and interindividual variation if one wants to highlight toxicological effects. Hence, in the effort to better understand the variability of biological responses, we must acquire a good understanding on the life cycle of the organism and seek the normal range of responses of the biomarker over time (seasonal variability) in a given age range [2]. However, the knowledge of all these aspects might be difficult when dealing with a new species not before examined. The understanding of the normal range of responses of biomarkers actually represents an important aspect of ecotoxicology research and

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