BIOASSAYS CONCEPTS, PRINCIPLES AND APPLICATION FOR PESTICIDES - PowerPoint PPT Presentation

 Is an assay designed to analyse any compound by use of a suitable biological system like animals, tissues, microbes etc  Is an estimation or determination of concentration or potency of a physical, chemical or biological substance (agent) by means of measuring and comparing the magnitude of the response of the test with that of standard over a suitable biological system under standard set of conditions

 In the analysis the response produced by the test compound is compared with that of the standard sample using a biological system

Principle of bioassay  The bioassay compares the test sample with the same Internationally applicable standard substance  It determines the quantity of test sample required to produce an equivalent biological response to that of standard substance  Standard samples are accepted by expert committee at international level and they represent fixed units of activity

Indications for Bioassay  Bioassays, as compared to other methods of assays (e.g. chemical or physical assay) are less accurate, less elaborate, more laborious, more troublesome and more expensive

Indications for Bioassay  Active principle is unknown  Active principle cannot be isolated  Chemical method is either  Not available  If available, too complex  Insensitive to low doses  Unknown Chemical composition  Chemical composition is different but with the same action

Principles of Bioassay  Active principle to be assayed should show the same measured response in all animal species  The degree of biological (pharmacological/toxicological) response produced should be reproducible under identical conditions  The reference standard must owe its activity to the principle for which the sample is being bioassayed

 Activity assayed should be the activity of interest  Individual variations must be minimised / accounted for  Bioassay might measure a different aspects of the same substance compared to chemical assay

Types of Bioassays  Two types of Bioassays  Quantal Assays: Direct endpoint  Elicits an „All or None‟ response in different animals  Calculation of LD50 in target test organism (mice, rats, fish, plants)  Graded Response Assays [mostly on tissues]  Graded responses to varying doses  Unknown dose response measured on same tissue

Graded Response Assay  In these assays, as the dose increases there is an equivalent rise in response  The potency is estimated by comparing the Test sample responses with the standard response curve  The graded dose response relationship relates the size of the response to the substance in a single biologic unit

 As the dose administered increases the pharmacological response also increases and eventually reaches a steady level called the ceiling effect where there will be on further increase in response even with an increase in dose

 The graded dose response curve is obtained by plotting a graph with dose on the X-axis and response on the Y-axis  It is usually sigmoid in shape however to be useful in bio assay the log dose response curve (almost a straight line is used)  Conc. of unknown = Threshold dose of standard/threshold dose of test x Conc. of standard

Quantal: End Point Assay  As the name indicates, the threshold dose of the sample required to elicit a complete or a particular pharmacological/toxicological effect is determined and compared with standard  The determination of LD50 (LD=Lethal dose) or ED50 (ED= effective dose) is done by this method

Methods of Bioassay con1  Graded Response Assays [ Direct comparison on same tissues]  Interpolation: Conc. of unknown is read from a standard plot of a log dose response curve of at least 4 sub maximal concentrations  Matching / Bracketing: Const dose bracketed with varying doses of standard till exact match is obtained  Used when test sample is too small  Inaccurate & margin of error difficult to estimate

 Multiple Point Assays  3 point assay [combines active principles of matching with interpolation]  4 point assay [combines active principles of matching with interpolation]

3 point assay [2+1 dose assay]  Fast & convenient  Procedure [Eg Ach bioassay]  Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solution  Select two std doses s1& s2 [ in 1:2 dose ratio] from linear part of LDR [ Let the corresponding response be S1, S2]  Choose a test dose t with a response T between S1 & S2

 Record 4 sets data [Latin square: Randomisation reduces error] as follows  s1 s2 t  t s1 s2  s2 t s1  s1 s2 t  Plot mean of S1, S2 and T against dose. Calculate  Log Potency ratio [ M ] = [ (T – S1) / (S2-S1) ] X log d [d = dose ratio]

4 point assay [2 +2 dose assay]  Procedure [Eg Ach bioassay]  Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solution  Select two std doses s1& s2 from linear part of LDR [ Let the corresponding response be S1, S2]  Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ; Also s2/s1 = t2/t1 = 2

 Record 4 data sets [Latin square: Randomisation reduces error]  s1 s2 t1 t2  s2 t1 t2 s1  t1 t2 s1 s2  t2 s1 s2 t1  Plot mean of S1, S2 and T1, T2 against dose. Calculate  Log Potency ratio [M] = [ (T1 – S1 + T2 – S2) / (S2-S1 + T2-T1) ] X log d [d = dose ratio]

Measurements  Dose-response relationships – quantifiable through graphical interpolation or statistical analysis  Effective concentrations (50) – useful for risk assessment  Biochemical response to stress – Haemoglobin production  Daphnia magna  Limnar manda (duck weed)  Chironomus tenstans

Advantages & Uses of bioassay  Determine the concentration and the potency of the sample  Used to standardize drugs, vaccines, toxins or poisons, disinfectants, antiseptics  Determine the specificity of a compound  Certain complex compounds like vitamin B-12 which can't be analysed by simple assay techniques can be effectively estimated by bioassays

 Sometimes the chemical composition of samples are different but have same biological activity  For samples where no other methods of assays are available

Types of Assays  Chemical Assays  Spectrophotometry,  Spectrofluorimetry  Chromatography  Immunoassays  Microbiological assays

Bioassay systems and techniques  The bioassay systems vary based on the biological system used  Animals (mouse, rat, guinea pig, rabbits etc)  Plant bioassay (using plant constituents to evaluate a sample like(haemolytic activity) microbiological  Cell based assay (using microbes like bacteria, fungi or cultured cells for anti biotic compound screening etc)

In vivo techniques  These techniques employ a living animal recommended for the purpose of assay  The techniques aims to study the biological effect or response of the compound under screening in a living system directly  Ex: By use of rodents, rabbits etc.

Ex vivo techniques  These techniques employ a tissue or cells of recommended living system to study the effect of compound under test in suitable conditions within the stipulated time of organ survival outside the body  Eg The methods described in the videos employ a living tissue of an animal in an apparatus to study the contractile effect of drugs

In vitro techniques  These techniques employ a cell culture of recommended biological system to study the effect of compound under standard condition not similar to that of living environment.  The cell culture survives by utilization of the nutrition in the media  Ex: use of stem cells, cell culture, microbes (bacteria) etc

Key points for bioassay  Do thorough literature search  Utilize, build on existing information  Identify the gaps  Use living biota to assess the impact  Use suite of ecologically relevant biota  Know the details of biota of your choice  Multi-disciplinary experts is essential

The purpose of bioassay  The purpose of bioassay is to ascertain the potency of a drug and hence it serves as the quantitative part of any screening procedure (Research).  Other purpose of bioassay is to standardize the preparation so that each contains the uniform specified pharmacological/toxicological activity  In this way, it serves as a pointer in the Commercial Production of drugs when chemical assays are not available or do not suffice.  From the clinical point of view, bioassay may help in the diagnosis of various conditions

Biomonitoring Bioindication Bioassay

Bio-indicators  A bio-indicator is an organism giving information on the environmental conditions of its habitat by its presence or absence and its behaviour  Thus effects at the physiological level are not included in this definition??  The indicator concept states that the continued presence of certain species is an indication of the existence of a unique set of acceptable environmental conditions, whereas its absence would indicate the lack of appropriate environmental conditions

 Ecological indicators are parameters describing the structure and functioning of ecosystems, for example species diversity, population dynamics and nutrient cycling rates

Bio-indicators  Organisms, chemical markers or biological processes whose change indicates the altered environmental conditions  While direct sampling provides information about the conditions at the time of sampling only, bio- indicators provide a time-integrated estimate of past environmental conditions

 Time scales can vary depending on the actual indicator chosen  They therefore serve to detect changes in the environment even when measurements are not available or are too variable  For example, a reduced abundance of large foraminifera (marine micro-organisms) or the darkening of coral pigmentation may indicate that a reef has been exposed to poor water quality for several weeks or months

 Bio-indicators can also provide information on the harmful effects of contaminants at biochemical, molecular and cellular levels and can act as an early warning system for larger-scale effects  For example, reduced photosynthesis in a plant or a coral may indicate stress from exposure to herbicides

BIOMARKERS

Analytical approach  Chemical analysis  Biomarkers

Definition  Biomarker  Any change that occurs in response to exposure to stressors (xenobiotics, disease causing agents and physical change in the environment including temperature and salinity) that indicates the adaptive responses of an organism beyond the normal state

 Examples  Induction of heat shock proteins which are triggered in response to raised temperatures  Reduction in fecundity – due to environmental pollutants  Plasma protein differentiating live and degenerated Taenia solium cysts

Biomarker concept 78  Biomarker  biological response measured to indicate exposure, effect/response or susceptibility  Biomarkers  Allow the identification of pollutants and their potential risks to the environment  Give additional information that cannot be obtained from chemical analysis  May show integrated effects of chemical mixtures 27.01.2014

Attributes of Biomarkers  Biomarkers are used to identify biological changes due to toxic chemicals and as part of integrated approach in the assessment of environmental health  They increase the ability to identify the long-term risks due to toxicant exposure in particular due to risks of developing cancer  The field of biomarkers identifies early markers of toxicity in the field of environmental toxicology or Ecotoxicology

 The ultimate aim of using biomarkers is to identify problems as early as possible, thus avoiding adverse effects on whole populations and communities  The goal of using biomarkers is to identify the adverse effects of chemical contaminants at the lowest level of biological organization so as to avoid toxicological problems at later stages which are both more difficult to identify and to correct  It is possible to identify biomarkers at all levels of biological organization extending from molecular of an individual to the ecosystem

Increased susceptibility Response to diseases Observable impaired Reduced detrimental reproduction lifespan effects No observable detrimental effects increased exposure Homeostasis (dose and time) Early warning signals normal range of biomarker responses of biomarkers The principle scheme of responses in organisms towards the detrimental effects of pollutant exposure 81

Effect Ef ect of of a agent gent exposur xposure a e at dif t differ erent le ent levels els Exposure of of biolo biologica gical l or organ ganiza ization tion Kinetics in tissues Binding to Seconds to minutes Receptors (mol) Minutes Biochemical to hours Responses (cell) Days to Physiological weeks Alterations (org) Weeks to Effect on months individuals Months to Effect on years Time to Present Ecological Easy of Pop, comm, complete status of relevance obtaining & ecosyst research knowledge of data data 82

Important criteria for biomarkers  Ideally, biomarkers should be:  The assay to quantify the biomarker should be sensitive, reliable and relatively cheap and easy to perform  The baseline data (concentration or activity) of the biomarker should be well defined in order to distinguish between natural variability (noise) and contaminant-induced stress (signal)  The basic biology and physiology of the organism should be known so that sources of uncontrolled variation (growth and development, reproduction, food sources) can be minimized.

 The confounding factors (intrinsic and extrinsic) to the biomarker response should be well established  It should be established whether changes in biomarker concentration are due to physiological acclimation or to genetic adaptation  The biomarker response should correlate with the „health‟ or „fitness‟ of the organism  It should preferably be non-invasive or non-destructive to allow or facilitate monitoring the effects of environmental pollution in protected or endangered species

Use of Biomarkers  The use of biomarkers measured at the molecular or cellular level has been proved to be of great value as a sensitive early warning tool for measuring biological effects in environmental quality assessments  The most compelling reason for using biomarkers is that they can give information on the biological effects of pollutants, rather than a mere quantification of environmental levels  Biomarkers applied both in laboratory and field studies can provide an important linkage between the laboratory toxicity and field-based assessments  Using field samples, biomarker data may provide an important index of the total external load that is biologically available in the exposure environment

 The merits of using biomarkers are summarized as follows:  They can demonstrate the interactions that have taken place between contaminants even at sub-lethal concentrations and effects in the organisms  They can detect the presence and/or effects of both known and unknown contaminants  They have the ability to allow early detection of effects from contaminants, thus providing an opportunity for remedial or preventive action to be taken, before irreversible environmental damage with ecological consequences occurs  They provide a temporal and spatial integrative measure of bioavailable pollutants

 They can help to establish the important routes of exposure if applied to species from different trophic levels and thus can aid in prioritizing monitoring schemes and strategies for intervention or remediation  They can detect exposure to, and the toxic effects of parent compounds and metabolites of readily metabolized and eliminated contaminants such as PAHs and organophosphates  They can integrate the toxicological interactions of mixtures of various pollutants and give an expression of cumulative effect at molecular, cellular or tissue targets

Classification  Three major groups that include:  Biomarkers of exposure  Biomarkers of effect or response  Biomarkers of susceptibility

Biomarkers of exposure  A biomarker of exposure refers to an exogenous substance or its metabolite, or the product of an interaction between a xenobiotic agent and some target molecule or cell, which is measured in a compartment within an organism  Biomarkers of exposure can be used to confirm and assess the exposure of an individual or population to a particular substance (group), by providing a link between external exposure and internal dosimetry  Biomarkers of exposure are divided into markers of internal dose and makers of effective dose

Markers of internal dose  Give an indication of the occurrence and extent of exposure of the organism and thus the likely concentration of the present compound or the metabolite at the target site  Markers of internal dose are useful in establishing the dose of a compound which has been absorbed in ecological studies and in human studies when they provide information about long-term carcinogen exposure

Markers of effective dose  Gives an indication of the true extent of the exposure of what is believed to be the target molecule, structure or cell  Chemicals can bind covalently to cellular macromolecules such as nucleic acids and proteins which may be the target molecule for the compound  These are called adducts and can be measured in tissues or body fluids

 Both markers are preferable to measuring external levels of the compound in question such as in workplace since they take into account the biological variations in absorption, metabolism and distribution of the compound in an individual  Due to many inter-individual differences in the rate and route of metabolism of a compound and also the accessibility of the target, any measurement of the internal dose and effective dose will be different  Hence, the effective dose at the target site is the preferred measurement to internal dose

Biomarkers of effect or response  These are biomarkers that have been used most widely and routinely  A biomarker of effect refers to a measurable biochemical, physiological or other alterations within an organism that, depending upon the magnitude, can be recognized to be associated with an established or possible health impairment or disease  Biomarkers of effect can be used to demonstrate either preclinical alterations or adverse health effects due to external exposure and absorption of a chemical  They can be grouped into several categorises such as metabolic, pathological, clinical, behavioural etc.

 A metabolic lesion may or may not be the result of altered pathology, rather it may predict or precipitate a pathological lesion, making them potential early warning markers such as elevated glucose levels in diabetic patients  There is also a growing interest in the use of and identification of non-invasive biomarkers rather than invasive biomarkers

 Non-invasive biomarkers allow routine sampling and also overcome the ethical issues  Thus biomarkers identified in urine, breath or saliva are more useful/preferred than those measured in blood

Behaviour and clinical markers  They are simplest biomarkers which are sometimes referred as gross indices  They include changes in body weight, urinary output, food consumption, population size and general behaviour  These changes may signify a change in the biochemistry or pathology of an individual  These changes may be the first indication that there is a problem in the environment  In toxicology trials, body weight can be very sensitive of adverse effects of a compound

Pathology  Invasive markers of tissue damage that cover an array of pathological techniques including gross pathology, organ weight and histology using light and electron microscopy  Measurement of enzyme change using immunohistochemistry

Clinical chemistry/pathology  Traditionally, fluids have been a source of biochemical markers which are able to identify both site and severity of a lesion within the organism  Elevate serum levels of enzyme which have leaked from the damaged tissue  Biochemical changes such as elevated bilirubin  Changes in biochemistry of urine and cerebrospinal fluid

Enzymatic changes (Induction /inhibition)  Changes in enzyme activity can be used as biomarkers of specific chemical exposure  Organophosphate exposure – inhibition of blood acetylcholinesterase  Lead exposure – inhibition of serum aminoaevulinic acid dehydrase (ALAD)  As these biomarkers are believed to bespecific, the degree of enzyme inhibition has also been used as biomarkers of “effective dose”

 Induction of specific enzymes e.g. cytochrome P450 isoenzymes is an adaptive response to challenges from a wide variety of compounds including organochlorines, polycyclic aromatic hydrocarbons  Direct measurements require tissue be sampled, although urinary markers of CYP P450 activity such as excretion of D- glucaric acid in urine can be used as a non-invasive marker  In the recent years it has been shown that the constituents of breath are also potential source of CYP P450 generated metabolites which should also be used as biomarkers