ES/RP 531 Fundamentals of Environmental Toxicology Fall 2003ESRP531 Lect 22 Biomar_[1].doc Page 1 of 23December 1, 2003Lecture 22: Biomarkers; Environmental and Tissue Contaminant ResiduesI. BiomarkersA. Biomarkers are biochemical, physiological, or histological indicators of eitherexposure to or effects of contaminants (or even natural products) at thesuborganismal or organismal level of organization.1. Biomarkers would be indicative of a contaminant effect or an exposure at theindividual level.2. However, biomarker quantification can be used to compare populations livingin polluted vs. “pristine” sites to gain insight into potential exposure to toxicsubstances among populations.B. Biomarkers are generally measurements of sublethal effects in that livingorganisms are collected and assayed. However, some biomarkers can bereasonably hypothesized to be indicative of a type of contaminant, and thusbiomarkers can be more specific measures of exposure.1. One example of specificity is the use of acetylcholinesterase (AChE) activity,which can be inhibited by insecticides of the organophosphorus (OP) andcarbamate (CB) classes;a. AChE can be measured in live or dead organisms (assuming they haven’tbeen so long that the tissue and therefore the enzymes has decomposed.b. However, caution must be used because other compounds can inhibitAChE, including natural products (e.g., certain alkaloids) and heavymetals (e.g., copper).C. Types of biomarkers1. Enzymesa. AChE—may be indicative of exposure to OP and CB insecticidesb. ALAD (delta-aminolevulinic acid dehydratase): indicative of leadexposurec. ATPased. Plant enzymes (peroxidase, RUBISCO)2. Energeticsa. Adenylate energy chargeb. Energy reservesc. Whole body Calorimetryd. Enzymes of intermediary metabolisme. Growth3. Endocrinea. Hormone levels1. Corticosteroids/catecholamines2. Thyroid hormone3. Estrogen/Testosterone4. Insulin/Glucagon5. Growth hormoneb. Protein synthesis under endocrine control1. Vitellogenin levels in male fishES/RP 531 Fundamentals of Environmental Toxicology Fall 2003ESRP531 Lect 22 Biomar_[1].doc Page 2 of 234. Blood chemistry5. Growth Ratea. RNA and protein synthesisD. Limitations of biomarkers1. Lack of specificity2. Natural (endogenous) variability of response or variability of response asrelated to other environmental stressorsa. As an example of variability among different populations, Olsen et al.(2001) ETAC 20:1725 showed that the biomarker enzymes, glutathione-s-transferase (GST) and acetylcholinesterase varied by factors of 1.84- and1.81-fold, respectively, in Chironomus larvae held under ambientconditions of 13 “uncontaminated sites” (see Figure 1).Figure 1. Mean GST and AChE (mM/L/min/g protein) in whole body of Chironomusriparius larvae deployed in situ for 48 h at 13 uncontaminated river sites in theU.K. (Olsen et al., 2001, Environ. Toxicol. Chem. 20:1725-1732.)II. Analysis of Contaminant Residues: Issues in Environmental Analytical ChemistryA. Although biomarkers can be indicative of exposure, they are often not specificenough to determine the identity of a chemical and therefore are difficult to use todetermine specific dosage (or concentration) to which an organism has beenexposed.B. Measurement of contaminant residues both in the environment (which is usefulfor predicting exposure) and in tissues (which allows an estimation of what anorganism has already been exposed to) is the most accurate and reliable way toES/RP 531 Fundamentals of Environmental Toxicology Fall 2003ESRP531 Lect 22 Biomar_[1].doc Page 3 of 23estimate exposure. Thus, environmental analytical chemistry is the tool of choicefor exposure estimation.1. In addition to their importance in estimating exposure, residues ofcontaminants are monitored for compliance with various numerical standards.a. For example, under the Clean Water Act, EPA can promulgate guidelinesknown as ambient water quality criteria for the protection of aquatic biota.1. These guidelines are not enforceable on the Federal level, but statescan adopt similar guidelines that may be enforceable standards.2. In some cases the standards, criteria, or guidelines for protection ofaquatic biota are as low as 1 part per trillion (i.e., 1 ppt--applies toDDT residues)2. Thus, knowing how residue numbers are produced and some of the issuesrelated to detection and quantitation makes us a bit more skeptical and perhapsappreciative of the tremendous amount of residue monitoring that is presentedin technical reports and the refereed scientific literature.C. The “Desperately Seeking Nothing” Syndrome1. Capability of analyzing contaminants to levels of ppt and even below hasevolved without a corresponding understanding of biological effects at theselevels (Figure 2).10-310010-610-910-121950 1960 1970 1980 1990 2000ppthppmppbpptppq?10-15g / LYearFigure 2. Evolution of analytical capability.a. Bear in mind that requirements for toxicological testing only demandcomparatively high dose testing; i.e., the doses where a biological effect islikely to be observed (although many contaminants will not produce abiological effect in chronic toxicity testing unless high doses relative toenvironmental concentrations are administered)2. What does it mean to analyze something to the level of ppt?a. Significance of substance purity by percentage1. 99.9999% purea. 0.0001% impurities or 1 ppm of impurities2. 99.9999999% purea. 0.0000001% impurities or 1 ppb of impurities3. 99.9999999999% purea. 0.0000000001% impurities or 1 ppt of impuritiesES/RP 531 Fundamentals of Environmental Toxicology Fall 2003ESRP531 Lect 22 Biomar_[1].doc Page 4 of 23b. On the other hand, 1 ppb of a pesticide with molecular weight of300 contains 2 x 1015 molecules per Liter, and 1 ppt in watercontains 2 x 1012 molecules1. It’s a lot of molecules but is it enough to react with an enzymeor other biological receptor to cause a measurable reaction?a. Remember the kinetic parameter, Km, used in enzymebiochemistry to indicate the affinity of a substrate andenzyme (Km = the concentration at which the reactionvelocity is 50% of maximal velocity)3. Consequences of Increased Analytical Capabilities (my opinion)a. Contaminants seem to occur in places that we had never seen them before1. For ex., based on the Koc of DDT, we would not have predicted itwould leach to ground water; yet, it has been found in ground water,albeit at levels of ppt.b. We have the